Methods for Treating Conditions Associated with MASP-2 Dependent Complement Activation

ABSTRACT

In one aspect, the invention provides methods of inhibiting the effects of MASP-2-dependent complement activation in a living subject suffering from, or at risk for developing a thrombotic microangiopathy (TMA). The methods comprise the step of administering, to a subject in need thereof, an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitory agent inhibits cellular injury associated with MASP-2-mediated alternative complement pathway activation, while leaving the classical (C1q-dependent) pathway component of the immune system intact.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Prior application Ser. No.14/517,750 filed Oct. 17, 2014, which claims the benefit of ProvisionalApplication No. 61/892,283, filed Oct. 17, 2013, now expired, and thisapplication claims the benefit of Provisional Application No.62/020,845, filed Jul. 3, 2014, now expired, each of which is herebyincorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing isMP_1_0220_US2_Sequence_Listing_20190711_ST25.txt. The text file is 115KB; was created on Jul. 11, 2019; and is being submitted via EFS-Webwith the filing of the specification.

BACKGROUND

The complement system provides an early acting mechanism to initiate,amplify and orchestrate the immune response to microbial infection andother acute insults (M. K. Liszewski and J. P. Atkinson, 1993, inFundamental Immunology, Third Edition, edited by W. E. Paul, RavenPress, Ltd., New York), in humans and other vertebrates. Whilecomplement activation provides a valuable first-line defense againstpotential pathogens, the activities of complement that promote aprotective immune response can also represent a potential threat to thehost (K. R. Kalli, et al., Springer Semin. Immunopathol. 15:417-431,1994; B. P. Morgan, Eur. J. Clinical Inveslig. 24:219-228, 1994). Forexample, C3 and C5 proteolytic products recruit and activateneutrophils. While indispensable for host defense, activated neutrophilsare indiscriminate in their release of destructive enzymes and may causeorgan damage. In addition, complement activation may cause thedeposition of lytic complement components on nearby host cells as wellas on microbial targets, resulting in host cell lysis.

The complement system has also been implicated in the pathogenesis ofnumerous acute and chronic disease states, including: myocardialinfarction, stroke, ARDS, reperfusion injury, septic shock, capillaryleakage following thermal burns, postcardiopulmonary bypassinflammation, transplant rejection, rheumatoid arthritis, multiplesclerosis, myasthenia gravis, and Alzheimer's disease. In almost all ofthese conditions, complement is not the cause but is one of severalfactors involved in pathogenesis. Nevertheless, complement activationmay be a major pathological mechanism and represents an effective pointfor clinical control in many of these disease states. The growingrecognition of the importance of complement-mediated tissue injury in avariety of disease states underscores the need for effective complementinhibitory drugs. To date, Eculizumab (Solaris®), an antibody againstC5, is the only complement-targeting drug that has been approved forhuman use. Yet, C5 is one of several effector molecules located“downstream” in the complement system, and blockade of C5 does notinhibit activation of the complement system. Therefore, an inhibitor ofthe initiation steps of complement activation would have significantadvantages over a “downstream” complement inhibitor.

Currently, it is widely accepted that the complement system can beactivated through three distinct pathways: the classical pathway, thelectin pathway, and the alternative pathway. The classical pathway isusually triggered by a complex composed of host antibodies bound to aforeign particle (i.e., an antigen) and thus requires prior exposure toan antigen for the generation of a specific antibody response. Sinceactivation of the classical pathway depends on a prior adaptive immuneresponse by the host, the classical pathway is part of the acquiredimmune system. In contrast, both the lectin and alternative pathways areindependent of adaptive immunity and are part of the innate immunesystem.

The activation of the complement system results in the sequentialactivation of serine protease zymogens. The first step in activation ofthe classical pathway is the binding of a specific recognition molecule,C1q, to antigen-bound IgG and IgM molecules. C1q is associated with theC1r and C1s serine protease proenzymes as a complex called C1. Uponbinding of C1q to an immune complex, autoproteolytic cleavage of theArg-Ile site of C1r is followed by C1r-mediated cleavage and activationof C1s, which thereby acquires the ability to cleave C4 and C2. C4 iscleaved into two fragments, designated C4a and C4b, and, similarly, C2is cleaved into C2a and C2b. C4b fragments are able to form covalentbonds with adjacent hydroxyl or amino groups and generate the C3convertase (C4b2a) through noncovalent interaction with the C2a fragmentof activated C2. C3 convertase (C4b2a) activates C3 by proteolyticcleavage into C3a and C3b subcomponents leading to generation of the C5convertase (C4b2a3b), which, by cleaving C5 leads to the formation ofthe membrane attack complex (C5b combined with C6, C7, C8 and C-9, alsoreferred to as “MAC”) that can disrupt cellular membranes leading tocell lysis. The activated forms of C3 and C4 (C3b and C4b) arecovalently deposited on the foreign target surfaces, which arerecognized by complement receptors on multiple phagocytes.

Independently, the first step in activation of the complement systemthrough the lectin pathway is also the binding of specific recognitionmolecules, which is followed by the activation of associated serineprotease proenzymes. However, rather than the binding of immunecomplexes by C1q, the recognition molecules in the lectin pathwaycomprise a group of carbohydrate-binding proteins (mannan-binding lectin(MBL), H-ficolin, M-ficolin, L-ficolin and C-type lectin CL-11),collectively referred to as lectins. See J. Lu et al., Biochim. Biophys.Acta 1572:387-400, (2002); Holmskov et al., Annu. Rev. Immunol.21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000)). See alsoJ. Luet et al., Biochim Biophys Acta 1572:387-400 (2002); Holmskov etal, Annu Rev Immunol 21:547-578 (2003); Teh et al., Immunology101:225-232 (2000); Hansen et al, J. Immunol 185(10):6096-6104 (2010).

Ikeda et al. first demonstrated that, like C1q, MBL could activate thecomplement system upon binding to yeast mannan-coated erythrocytes in aC4-dependent manner (Ikeda et al., J. Biol. Chem. 262:7451-7454,(1987)). MBL, a member of the collectin protein family, is acalcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxygroups oriented in the equatorial plane of the pyranose ring. Prominentligands for MBL are thus D-mannose and N-acetyl-D-glucosamine, whilecarbohydrates not fitting this steric requirement have undetectableaffinity for MBL (Weis et al., Nature 360:127-134, (1992)). Theinteraction between MBL and monovalent sugars is extremely weak, withdissociation constants typically in the single-digit millimolar range.MBL achieves tight, specific binding to glycan ligands by avidity, i.e.,by interacting simultaneously with multiple monosaccharide residueslocated in close proximity to each other (Lee et al., Archiv. Biochem.Biophys. 299:129-136, (1992)). MBL recognizes the carbohydrate patternsthat commonly decorate microorganisms such as bacteria, yeast, parasitesand certain viruses. In contrast, MBL does not recognize D-galactose andsialic acid, the penultimate and ultimate sugars that usually decorate“mature” complex glycoconjugates present on mammalian plasma and cellsurface glycoproteins. This binding specificity is thought to promoterecognition of “foreign” surfaces and help protect from“self-activation.” However, MBL does bind with high affinity to clustersof high-mannose “precursor” glycans on N-linked glycoproteins andglycolipids sequestered in the endoplasmic reticulum and Golgi ofmammalian cells (Maynard et al., J. Biol. Chem. 257:3788-3794, (1982)).Therefore, damaged cells are potential targets for lectin pathwayactivation via MBL binding.

The ficolins possess a different type of lectin domain than MBL, calledthe fibrinogen-like domain. Ficolins bind sugar residues in aCa⁺⁺-independent manner. In humans, three kinds of ficolins (L-ficolin,M-ficolin and H-ficolin) have been identified. The two serum ficolins,L-ficolin and H-ficolin, have in common a specificity forN-acetyl-D-glucosamine; however, H-ficolin also bindsN-acetyl-D-galactosamine. The difference in sugar specificity ofL-ficolin, H-ficolin, CL-11, and MBL means that the different lectinsmay be complementary and target different, though overlapping,glycoconjugates. This concept is supported by the recent report that, ofthe known lectins in the lectin pathway, only L-ficolin bindsspecifically to lipoteichoic acid, a cell wall glycoconjugate found onall Gram-positive bacteria (Lynch et al., J. Immunol. 172:1198-1202,(2004)). The collectins (i.e., MBL) and the ficolins bear no significantsimilarity in amino acid sequence. However, the two groups of proteinshave similar domain organizations and, like Clq, assemble intooligomeric structures, which maximize the possibility of multisitebinding.

The serum concentrations of MBL are highly variable in healthypopulations and this is genetically controlled bypolymorphisms/mutations in both the promoter and coding regions of theMBL gene. As an acute phase protein, the expression of MBL is furtherupregulated during inflammation. L-ficolin is present in serum atconcentrations similar to those of MBL. Therefore, the L-ficolin branchof the lectin pathway is potentially comparable to the MBL arm instrength. MBL and ficolins can also function as opsonins, which allowphagocytes to target MBL- and ficolin-decorated surfaces (see Jack etal., J Leukoc Biol., 77(3):328-36 (2004), Matsushita and Fujita,Immunobiology, 205(4-5):490-7 (2002), Aoyagi et al., J Immunol,174(1):418-25(2005). This opsonization requires the interaction of theseproteins with phagocyte receptors (Kuhlman et al., J. Exp. Med.169:1733, (1989); Matsushita et al., J. Biol. Chem. 271:2448-54,(1996)), the indentity of which has not been established.

Human MBL forms a specific and high-affinity interaction through itscollagen-like domain with unique Clr/Cls-like serine proteases, termedMBL-associated serine proteases (MASPs). To date, three MASPs have beendescribed. First, a single enzyme “MASP” was identified andcharacterized as the enzyme responsible for the initiation of thecomplement cascade (i.e., cleaving C2 and C4) (Matsushita et al., J ExpMed 176(6):1497-1502 (1992); Ji et al., J. Immunol. 150:571-578,(1993)). It was subsequently determined that the MASP activity was, infact, a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al.,Nature 386:506-510, (1997)). However, it was demonstrated that theMBL-MASP-2 complex alone is sufficient for complement activation(Vorup-Jensen et al., J. Immunol. 165:2093-2100, (2000)). Furthermore,only MASP-2 cleaved C2 and C4 at high rates (Ambrus et al., J. Immunol.170:1374-1382, (2003)). Therefore, MASP-2 is the protease responsiblefor activating C4 and C2 to generate the C3 convertase, C4b2a. This is asignificant difference from the C1 complex of the classical pathway,where the coordinated action of two specific serine proteases (C1r andC1s) leads to the activation of the complement system. In addition, athird novel protease, MASP-3, has been isolated (Dahl, M. R., et al.,Immunity 15:127-35, 2001). MASP-1 and MASP-3 are alternatively splicedproducts of the same gene.

MASPs share identical domain organizations with those of C1r and C1s,the enzymatic components of the C1 complex (Sim et al., Biochem. Soc.Trans. 28:545, (2000)). These domains include an N-terminal C1r/C1s/seaurchin VEGF/bone morphogenic protein (CUB) domain, an epidermal growthfactor-like domain, a second CUB domain, a tandem of complement controlprotein domains, and a serine protease domain. As in the C1 proteases,activation of MASP-2 occurs through cleavage of an Arg-Ile bond adjacentto the serine protease domain, which splits the enzyme intodisulfide-linked A and B chains, the latter consisting of the serineprotease domain.

MBL can also associate with an alternatively sliced form of MASP-2,known as MBL-associated protein of 19 kDa (MAp19) or smallMBL-associated protein (sMAP), which lacks the catalytic acivity ofMASP2. (Stover, J. Immunol. 162:3481-90, (1999); Takahashi et al., Int.Immunol. 11:859-863, (1999)). MAp19 comprises the first two domains ofMASP-2, followed by an extra sequence of four unique amino acids. Thefunction of Map19 is unclear (Degn et al., JImmunol. Methods, 2011). TheMASP-1 and MASP-2 genes are located on human chromosomes 3 and 1,respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)).

Several lines of evidence suggest that there are different MBL-MASPcomplexes and a large fraction of the MASPs in serum is not complexedwith MBL (Thiel, et al., J. Immunol. 165:878-887, (2000)). Both H- andL-ficolin bind to all MASPs and activate the lectin complement pathway,as does MBL (Dahl et al., Immunity 15:127-35, (2001); Matsushita et al.,J. Immunol. 168:3502-3506, (2002)). Both the lectin and classicalpathways form a common C3 convertase (C4b2a) and the two pathwaysconverge at this step.

The lectin pathway is widely thought to have a major role in hostdefense against infection in the naïve host. Strong evidence for theinvolvement of MBL in host defense comes from analysis of patients withdecreased serum levels of functional MBL (Kilpatrick, Biochim. Biophys.Acta 1572:401-413, (2002)). Such patients display susceptibility torecurrent bacterial and fungal infections. These symptoms are usuallyevident early in life, during an apparent window of vulnerability asmaternally derived antibody titer wanes, but before a full repertoire ofantibody responses develops. This syndrome often results from mutationsat several sites in the collagenous portion of MBL, which interfere withproper formation of MBL oligomers. However, since MBL can function as anopsonin independent of complement, it is not known to what extent theincreased susceptibility to infection is due to impaired complementactivation.

In contrast to the classical and lectin pathways, no initiators of thealternative pathway have been found to fulfill the recognition functionsthat C1q and lectins perform in the other two pathways. Currently it iswidely accepted that the alternative pathway spontaneously undergoes alow level of turnover activation, which can be readily amplified onforeign or other abnormal surfaces (bacteria, yeast, virally infectedcells, or damaged tissue) that lack the proper molecular elements thatkeep spontaneous complement activation in check. There are four plasmaproteins directly involved in the activation of the alternative pathway:C3, factors B and D, and properdin.

Although there is extensive evidence implicating both the classical andalternative complement pathways in the pathogenesis of non-infectioushuman diseases, the role of the lectin pathway is just beginning to beevaluated. Recent studies provide evidence that activation of the lectinpathway can be responsible for complement activation and relatedinflammation in ischemia/reperfusion injury. Collard et al. (2000)reported that cultured endothelial cells subjected to oxidative stressbind MBL and show deposition of C3 upon exposure to human serum (Collardet al., Am. J. Pathol. 156:1549-1556, (2000)). In addition, treatment ofhuman sera with blocking anti-MBL monoclonal antibodies inhibited MBLbinding and complement activation. These findings were extended to a ratmodel of myocardial ischemia-reperfusion in which rats treated with ablocking antibody directed against rat MBL showed significantly lessmyocardial damage upon occlusion of a coronary artery than rats treatedwith a control antibody (Jordan et al., Circulation 104:1413-1418,(2001)). The molecular mechanism of MBL binding to the vascularendothelium after oxidative stress is unclear; a recent study suggeststhat activation of the lectin pathway after oxidative stress may bemediated by MBL binding to vascular endothelial cytokeratins, and not toglycoconjugates (Collard et al., Am. J. Pathol. 159:1045-1054, (2001)).Other studies have implicated the classical and alternative pathways inthe pathogenesis of ischemia/reperfusion injury and the role of thelectin pathway in this disease remains controversial (Riedermann, N.C.,et al., Am. J. Pathol. 162:363-367, 2003).

A recent study has shown that MASP-1 (and possibly also MASP-3) isrequired to convert the alternative pathway activation enzyme Factor Dfrom its zymogen form into its enzymatically active form (see TakahashiM. et al., J Exp Med 207(1):29-37 (2010)). The physiological importanceof this process is underlined by the absence of alternative pathwayfunctional activity in plasma of MASP-1/3-deficient mice. Proteolyticgeneration of C3b from native C3 is required for the alternative pathwayto function. Since the alternative pathway C3 convertase (C3bBb)contains C3b as an essential subunit, the question regarding the originof the first C3b via the alternative pathway has presented a puzzlingproblem and has stimulated considerable research.

C3 belongs to a family of proteins (along with C4 and α-2 macroglobulin)that contain a rare posttranslational modification known as a thioesterbond. The thioester group is composed of a glutamine whose terminalcarbonyl group forms a covalent thioester linkage with the sulfhydrylgroup of a cysteine three amino acids away. This bond is unstable andthe electrophilic glutamyl-thioester can react with nucleophilicmoieties such as hydroxyl or amino groups and thus form a covalent bondwith other molecules. The thioester bond is reasonably stable whensequestered within a hydrophobic pocket of intact C3. However,proteolytic cleavage of C3 to C3a and C3b results in exposure of thehighly reactive thioester bond on C3b and, following nucleophilic attackby adjacent moieties comprising hydroxyl or amino groups, C3b becomescovalently linked to a target. In addition to its well-documented rolein covalent attachment of C3b to complement targets, the C3 thioester isalso thought to have a pivotal role in triggering the alternativepathway. According to the widely accepted “tick-over theory”, thealternative pathway is initiated by the generation of a fluid-phaseconvertase, iC3Bb, which is formed from C3 with hydrolyzed thioester(iC3; C3(H₂O)) and factor B (Lachmann, P. J., et al., Springer Semin.Immunopathol. 7:143-162, (1984)). The C3b-like C3(H₂O) is generated fromnative C3 by a slow spontaneous hydrolysis of the internal thioester inthe protein (Pangburn, M. K., et al., J. Exp. Med. 154:856-867, 1981).Through the activity of the C3(H₂O)Bb convertase, C3b molecules aredeposited on the target surface thereby initiating the alternativepathway.

Very little is known about the initiators of activation of thealternative pathway. Activators are thought to include yeast cell walls(zymosan), many pure polysaccharides, rabbit erythrocytes, certainimmunoglobulins, viruses, fungi, bacteria, animal tumor cells,parasites, and damaged cells. The only feature common to theseactivators is the presence of carbohydrate, but the complexity andvariety of carbohydrate structures has made it difficult to establishthe shared molecular determinants which are recognized. It has beenwidely accepted that alternative pathway activation is controlledthrough the fine balance between inhibitory regulatory components ofthis pathway, such as Factor H, Factor I, DAF, and CR1, and properdin,which is the only positive regulator of the alternative pathway (seeSchwaeble W. J. and Reid K. B., Immunol Today 20(1):17-21 (1999)).

In addition to the apparently unregulated activation mechanism describedabove, the alternative pathway can also provide a powerful amplificationloop for the lectin/classical pathway C3 convertase (C4b2a) since anyC3b generated can participate with factor B in forming additionalalternative pathway C3 convertase (C3bBb). The alternative pathway C3convertase is stabilized by the binding of properdin. Properdin extendsthe alternative pathway C3 convertase half-life six to ten fold.Addition of C3b to the alternative pathway C3 convertase leads to theformation of the alternative pathway C5 convertase.

All three pathways (i.e., the classical, lectin and alternative) havebeen thought to converge at C5, which is cleaved to form products withmultiple proinflammatory effects. The converged pathway has beenreferred to as the terminal complement pathway. C5a is the most potentanaphylatoxin, inducing alterations in smooth muscle and vascular tone,as well as vascular permeability. It is also a powerful chemotaxin andactivator of both neutrophils and monocytes. C5a-mediated cellularactivation can significantly amplify inflammatory responses by inducingthe release of multiple additional inflammatory mediators, includingcytokines, hydrolytic enzymes, arachidonic acid metabolites, andreactive oxygen species. C5 cleavage leads to the formation of C5b-9,also known as the membrane attack complex (MAC). There is now strongevidence that sublytic MAC deposition may play an important role ininflammation in addition to its role as a lytic pore-forming complex.

In addition to its essential role in immune defense, the complementsystem contributes to tissue damage in many clinical conditions. Thus,there is a pressing need to develop therapeutically effective complementinhibitors to prevent these adverse effects.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one aspect, the present invention provides a method of inhibitingMASP-2-dependent complement activation in a subject suffering from, orat risk for developing a thrombotic microangiopathy (TMA), wherein theTMA is at least one of (i) a TMA secondary to cancer; (ii) a TMAsecondary to chemotherapy, or (iii) a TMA secondary to transplantation,comprising administering to the subject a composition comprising anamount of a MASP-2 inhibitory agent effective to inhibitMASP-2-dependent complement activation. In some embodiments, the subjectis suffering from, or is at risk for developing a TMA secondary tocancer, and the MASP-2 inhibitory agent is administered systemically tothe subject in an amount effective to reduce the risk of developing TMA,or reduce the severity of TMA. In some embodiments, the subject issuffering from, or is at risk for developing a TMA secondary tochemotherapy, and the MASP-2 inhibitory agent is administeredsystemically to the subject prior to, during, or after chemotherapy, inan amount effective to reduce the risk of developing TMA, or reduce theseverity of TMA. In some embodiments, the subject is suffering from, oris at risk for developing a TMA secondary to transplantation and theMASP-2 inhibitory agent is administered systemically to the subjectprior to, during, or after the transplant procedure, in an amounteffective to reduce the risk of developing TMA, or reduce the severityof TMA. In some embodiments the transplant procedure is an allogeneichematopoietic stem cell transplant. In some embodiments, the subject haspreviously undergone, or is currently undergoing, treatment with aterminal complement inhibitor that inhibits cleavage of complementprotein C5. In some embodiments, the method further comprisesadministering to the subject a terminal complement inhibitor thatinhibits cleavage of complement protein C5, such as a humanized anti-C5antibody or antigen-binding fragment thereof, such as eculizumab.

In another aspect, the invention provides a method of inhibitingMASP-2-dependent complement activation in a subject suffering from or atrisk for developing Upshaw-Schulman Syndrome (USS) comprisingadministering to the subject a composition comprising an amount of aMASP-2 inhibitory agent effective to inhibit MASP-2 dependent complementactivation. In some embodiments, the method comprises treating a subjectat risk for developing USS, wherein the method comprises administeringan amount of a MASP-2 inhibitory agent for a time period effective toameliorate or prevent one of more clinical symptoms associated with TTP.In some embodiments, the method further comprises periodicallymonitoring the subject and administering the MASP-2 inhibitory agentupon the presence of an event known to be associated with triggering TTPclinical symptoms. In some embodiments, the method further comprisesperiodically monitoring the subject and administering the MASP-2inhibitory agent upon the determination of the presence of anemia,thrombocytopenia or rising creatine. In some embodiments, the subjecthas previously undergone, or is currently undergoing, treatment with aterminal complement inhibitor that inhibits cleavage of complementprotein C5. In some embodiments, the method further comprisesadministering to the subject a terminal complement inhibitor thatinhibits cleavage of complement protein C5, such as a humanized anti-C5antibody or antigen-binding fragment thereof, such as eculizumab.

In another aspect, the invention provides a method of inhibitingMASP-2-dependent complement activation in a subject suffering from Degosdisease, comprising administering to the subject a compositioncomprising an amount of a MASP-2 inhibitory agent effective to inhibitMASP-2-dependent complement activation. In some embodiments, the subjecthas previously undergone, or is currently undergoing, treatment with aterminal complement inhibitor that inhibits cleavage of complementprotein C5. In some embodiments, the method further comprisesadministering to the subject a terminal complement inhibitor thatinhibits cleavage of complement protein C5, such as a humanized anti-C5antibody or antigen-binding fragment thereof, such as eculizumab.

In another aspect, the invention provides a method of inhibitingMASP-2-dependent complement activation in a subject suffering fromCatastrophic Antiphospholipid Syndrome (CAPS), comprising administeringto the subject a composition comprising an amount of a MASP-2 inhibitoryagent effective to inhibit MASP-2-dependent complement activation. Insome embodiments, the subject has previously undergone, or is currentlyundergoing, treatment with a terminal complement inhibitor that inhibitscleavage of complement protein C5. In some embodiments, the methodfurther comprises administering to the subject a terminal complementinhibitor that inhibits cleavage of complement protein C5, such as ahumanized anti-C5 antibody or antigen-binding fragment thereof, such aseculizumab.

In some embodiments of any of the disclosed methods of the invention,the MASP-2 inhibitory agent is a MASP-2 inhibitory antibody or fragmentthereof. In some embodiments, the MASP-2 inhibitory antibody has reducedeffector function. In some embodiments, the MASP-2 inhibitory antibodydoes not substantially inhibit the classical pathway. In someembodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonalantibody, or fragment thereof that specifically binds to a portion ofSEQ ID NO:6. In some embodiments, the anti-MASP-2 antibody or fragmentthereof is selected from the group consisting of a recombinant antibody,an antibody having reduced effector function, a chimeric antibody, ahumanized antibody and a human antibody. In some embodiments, the MASP-2inhibitory antibody is an antibody fragment selected from the groupconsisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)2. In some embodiments,the MASP-2 inhibitory antibody is a single-chain molecule. In someembodiments, the MASP-2 inhibitory antibody is selected from the groupconsisting of an IgG1 molecule, an IgG2 and an IgG4 molecule. In someembodiments, the MASP-2 inhibitory antibody is an IgG4 moleculecomprising a S228P mutation. In some embodiments, the MASP-2 inhibitoryantibody binds human MASP-2 with a K_(D) of 10 nM or less. In someembodiments, the MASP-2 inhibitory antibody binds an epitope in the CCP1domain of MASP-2. In some embodiments, the MASP-2 inhibitory antibodyinhibits C3b deposition in an in vitro assay in 1% human serum at anIC₅₀ of 10 nM or less. In some embodiments, the MASP-2 inhibitoryantibody inhibits C3b deposition in 90% human serum with an IC₅₀ of 30nM or less.

In some embodiments of any of the disclosed methods of the invention theMASP-2 inhibitory monoclonal antibody, or antigen-binding fragmentthereof, comprises: (a) a heavy-chain variable region comprising: i) aheavy chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acidsequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and (b) alight-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70. In some embodiments, the MASP-2inhibitory monoclonal antibody comprises a heavy-chain variable regionset forth as SEQ ID NO:67 and a light-chain variable region set forth asSEQ ID NO:70. In some embodiments, the MASP-2 inhibitory antibody orantigen binding-fragment thereof specifically recognizes at least partof an epitope recognized by a reference antibody comprising a heavychain variable region as set forth in SEQ ID NO:67 and a light-chainvariable region as set forth in SEQ ID NO:70.

In another aspect of the invention, methods are provided for inhibitingthrombus formation in a subject suffering from atypical hemolytic uremicsyndrome (aHUS), comprising administering to the subject an amount of aMASP-2 inhibitory antibody, or antigen binding fragment thereof,effective to inhibit MASP-2-dependent complement activation. In someembodiments, the MASP-2 inhibitory antibody inhibits thrombus formationin serum from a subject suffering from aHUS by at least 40% as comparedto untreated serum. In some embodiments, the MASP-2 inhibitory antibodyinhibits thrombus formation in serum from a subject suffering from aHUSat a level of at least 20% greater (e.g., at least 30% greater, at least40% greater, or at least 50% greater) than its inhibitory effect onC5b-9 deposition in the serum from the same subject. In someembodiments, the subject is in the acute phase of aHUS. In someembodiments, the subject is in the remission phase of aHUS. In someembodiments, the MASP-2 inhibitory antibody is a monoclonal antibody, orfragment thereof that specifically binds to a portion of SEQ ID NO:6. Insome embodiments, the MASP-2 inhibitory antibody or fragment thereof isselected from the group consisting of a recombinant antibody, anantibody having reduced effector function, a chimeric antibody, ahumanized antibody and a human antibody. In some embodiments, the MASP-2inhibitory antibody is an antibody fragment selected from the groupconsisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)₂. In some embodiments,the MASP-2 inhibitory antibody is a single-chain molecule. In someembodiments, the MASP-2 inhibitory antibody is selected from the groupconsisting of an IgG1 molecule, an IgG2 and an IgG4 molecule. In someembodiments, the MASP-2 inhibitory antibody is an IgG4 moleculecomprising a S228P mutation. In some embodiments, the MASP-2 inhibitoryantibody binds human MASP-2 with a K_(D) of 10 nM or less. In someembodiments, the MASP-2 inhibitory antibody binds an epitope in the CCP1domain of MASP-2. In some embodiments, the MASP-2 inhibitory antibodyinhibits C3b deposition in an in vitro assay in 1% human serum at anIC₅₀ of 10 nM or less. In some embodiments, the MASP-2 inhibitoryantibody inhibits C3b deposition in 90% human serum with an IC₅₀ of 30nM or less. In some embodiments the MASP-2 inhibitory monoclonalantibody, or antigen-binding fragment thereof, comprises: (a) aheavy-chain variable region comprising: i) a heavy chain CDR-H1comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) aheavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acidsequence from 95-102 of SEQ ID NO:67 and (b) a light-chain variableregion comprising: i) a light-chain CDR-L1 comprising the amino acidsequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii)a light-chain CDR-L3 comprising the amino acid sequence from 89-97 ofSEQ ID NO:70. In some embodiments, the MASP-2 inhibitory monoclonalantibody comprises a heavy-chain variable region set forth as SEQ IDNO:67 and a light-chain variable region set forth as SEQ ID NO:70. Insome embodiments, the MASP-2 inhibitory antibody or antigenbinding-fragment thereof specifically recognizes at least part of anepitope recognized by a reference antibody comprising a heavy chainvariable region as set forth in SEQ ID NO:67 and a light-chain variableregion as set forth in SEQ ID NO:70.

In another aspect, the present invention provides compositions forinhibiting the adverse effects of MASP-2-dependent complementactivation, comprising a therapeutically effective amount of a MASP-2inhibitory agent, such as a MASP-2 inhibitory antibody and apharmaceutically acceptable carrier. Methods are also provided formanufacturing a medicament for use in inhibiting the adverse effects ofMASP-2-dependent complement activation in living subjects in needthereof, comprising a therapeutically effective amount of a MASP-2inhibitory agent in a pharmaceutical carrier. Methods are also providedfor manufacturing medicaments for use in inhibiting MASP-2-dependentcomplement activation for treatment of each of the conditions, diseasesand disorders described herein below.

The methods, compositions and medicaments of the invention are usefulfor inhibiting the adverse effects of MASP-2-dependent complementactivation in vivo in mammalian subjects, including humans sufferingfrom or at risk for developing a thrombotic microangiopathy (TMA) asfurther described herein.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a diagram illustrating the genomic structure of human MASP-2;

FIG. 2A is a schematic diagram illustrating the domain structure ofhuman MASP-2 protein;

FIG. 2B is a schematic diagram illustrating the domain structure ofhuman MAp19 protein;

FIG. 3 is a diagram illustrating the murine MASP-2 knockout strategy;

FIG. 4 is a diagram illustrating the human MASP-2 minigene construct;

FIG. 5A presents results demonstrating that MASP-2-deficiency leads tothe loss of lectin-pathway-mediated C4 activation as measured by lack ofC4b deposition on mannan, as described in Example 2;

FIG. 5B presents results demonstrating that MASP-2-deficiency leads tothe loss of lectin-pathway-mediated C4 activation as measured by lack ofC4b deposition on zymosan, as described in Example 2;

FIG. 5C presents results demonstrating the relative C4 activation levelsof serum samples obtained from MASP-2+/−; MASP-2−/− and wild-typestrains as measure by C4b deposition on mannan and on zymosan, asdescribed in Example 2;

FIG. 6 presents results demonstrating that the addition of murinerecombinant MASP-2 to MASP-2−/− serum samples recoverslectin-pathway-mediated C4 activation in a protein concentrationdependant manner, as measured by C4b deposition on mannan, as describedin Example 2;

FIG. 7 presents results demonstrating that the classical pathway isfunctional in the MASP-2−/− strain, as described in Example 8;

FIG. 8A presents results demonstrating that anti-MASP-2 Fab2 antibody#11 inhibits C3 convertase formation, as described in Example 10;

FIG. 8B presents results demonstrating that anti-MASP-2 Fab2 antibody#11 binds to native rat MASP-2, as described in Example 10;

FIG. 8C presents results demonstrating that anti-MASP-2 Fab2 antibody#41 inhibits C4 cleavage, as described in Example 10;

FIG. 9 presents results demonstrating that all of the anti-MASP-2 Fab2antibodies tested that inhibited C3 convertase formation also were foundto inhibit C4 cleavage, as described in Example 10;

FIG. 10 is a diagram illustrating the recombinant polypeptides derivedfrom rat MASP-2 that were used for epitope mapping of the anti-MASP-2blocking Fab2 antibodies, as described in Example 11;

FIG. 11 presents results demonstrating the binding of anti-MASP-2 Fab2#40 and #60 to rat MASP-2 polypeptides, as described in Example 11;

FIG. 12 presents results demonstrating the blood urea nitrogen clearancefor wild type (+/+) and MASP-2 (−/−) mice at 24 and 48 hours afterreperfusion in a renal ischemia/reperfusion injury model, as describedin Example 12;

FIG. 13A presents results showing the baseline VEGF protein levels inRPE-choroid complex isolated from wild type (+/+) and MASP-2 (−/−) mice,as described in Example 13;

FIG. 13B presents results showing the VEGF protein levels in RPE-choroidcomplex at day 3 in wild type (+/+) and MASP-2 (−/−) mice followinglaser induced injury in a macular degeneration model, as described inExample 13;

FIG. 14 presents results showing the mean choroidal neovascularization(CNV) volume at day seven following laser induced injury in wild type(+/+) and MASP-2 (−/−) mice, as described in Example 13;

FIGS. 15A and 15B present dose response curves for the inhibition of C4bdeposition (FIG. 15A) and the inhibition of thrombin activation (FIG.15B) following the administration of a MASP-2 Fab2 antibody in normalrat serum, as described in Example 14;

FIGS. 16A and 16B present measured platelet aggregation (expressed asaggregate area) in MASP-2 (−/−) mice (FIG. 16B) as compared to plateletaggregation in untreated wild type mice and wild type mice in which thecomplement pathway is inhibited by depletory agent cobra venom factor(CVF) and a terminal pathway inhibitor (C5aR antagonist) (FIG. 16A) in alocalized Schwartzman reaction model of disseminated intravascularcoagulation, as described in Example 15;

FIG. 17 graphically illustrates the blood urea nitrogen (BUN) levelsmeasured in either WT (+/+) (B6) or MASP-2 (−/−) transplant recipientmice of WT (+/+) donor kidneys, as described in Example 16;

FIG. 18 graphically illustrates the percentage survival of WT (+/+) andMASP-2 (−/−) mice as a function of the number of days after microbialinfection in the cecal ligation and puncture (CLP) model, as describedin Example 17;

FIG. 19 graphically illustrates the number of bacteria measured in WT(+/+) and MASP-2 (−/−) after microbial infection in the cecal ligationand puncture (CLP) model, as described in Example 17;

FIG. 20 is a Kaplan-Mayer plot illustrating the percent survival of WT(+/+), MASP-2 (−/−) and C3 (−/−) mice six days after challenge withintranasal administration of Pseudomonas aeruginosa, as described inExample 18;

FIG. 21 graphically illustrates the level of C4b deposition, measured as% of control, in samples taken at various time points after subcutaneousdosing of either 0.3 mg/kg or 1.0 mg/kg of mouse anti-MASP-2 monoclonalantibody in WT mice, as described in Example 19;

FIG. 22 graphically illustrates the level of C4b deposition, measured as% of control, in samples taken at various time points after ip dosing of0.6 mg/kg of mouse anti-MASP-2 monoclonal antibody in WT mice, asdescribed in Example 19;

FIG. 23 graphically illustrates the mean choroidal neovascularization(CNV) volume at day seven following laser induced injury in WT (+/+)mice pre-treated with a single ip injection of 0.3 mg/kg or 1.0 mg/kgmouse anti-MASP-2 monoclonal antibody; as described in Example 20;

FIG. 24A graphically illustrates the percent survival of MASP-2 (−/−)and WT (+/+) mice after infection with 5×10⁸/100 μl cfu N. meningitidis,as described in Example 21;

FIG. 24B graphically illustrates the log cfu/ml of N. meningitidisrecovered at different time points in blood samples taken from theMASP-2 KO (−/−) and WT (+/+) mice infected with 5×10⁸ cfu/100 μl N.meningitidis, as described in Example 21;

FIG. 25A graphically illustrates the percent survival of MASP-2 KO (−/−)and WT (+/+) mice after infection with 2×10⁸ cfu/100 μl N. meningitidis,as described in Example 21;

FIG. 25B graphically illustrates the log cfu/ml of N. meningitidisrecovered at different time points in blood samples taken from the WT(+/+) mice infected with 2×10⁸ cfu/100 μl N. meningitidis, as describedin Example 21;

FIG. 25C graphically illustrates the log cfu/ml of N. meningitidisrecovered at different time points in blood samples taken from theMASP-2 (−/−) mice infected with 2×10⁸ cfu/100 μl N. meningitidis, asdescribed in Example 21;

FIG. 26A graphically illustrates the results of a C3b deposition assaydemonstrating that MASP-2 (−/−) mice retain a functional classicalpathway, as described in Example 22;

FIG. 26B graphically illustrates the results of a C3b deposition assayon zymosan coated plates, demonstrating that MASP-2 (−/−) mice retain afunctional alternative pathway, as described in Example 22;

FIG. 27A graphically illustrates myocardial ischemia/reperfusion injury(MIRI)-induced tissue loss following ligation of the left anteriordescending branch of the coronary artery (LAD) and reperfusion in C4(−/−) mice (n=6) and matching WT littermate controls (n=7), showing areaat risk (AAR) and infarct size (INF) as described in Example 22;

FIG. 27B graphically illustrates infarct size (INF) as a function ofarea at risk (AAR) in C4 (−/−) and WT mice treated as describe in FIG.42A, demonstrating that C4 (−/−) mice are as susceptible to MIRI as WTcontrols (dashed line), as described in Example 22;

FIG. 28A graphically illustrates the results of a C3b deposition assayusing serum from WT mice, C4 (−/−) mice and serum from C4 (−/−) micepre-incubated with mannan, as described in Example 22;

FIG. 28B graphically illustrates the results of a C3b deposition assayon serum from WT, C4 (−/−), and MASP-2 (−/−) mice mixed with variousconcentrations of an anti-murine MASP-2 mAb (mAbM 11), as described inExample 22;

FIG. 28C graphically illustrates the results of a C3b deposition assayon human serum from WT (C4 sufficient) and C4 deficient serum, and serumfrom C4 deficient subjects pre-incubated with mannan, as described inExample 22;

FIG. 28D graphically illustrates the results of a C3b deposition assayon human serum from WT (C4 sufficient) and C4 deficient subjects mixedwith anti-human MASP-2 mAb (mAbH3), as described in Example 22;

FIG. 29A graphically illustrates a comparative analysis of C3 convertaseactivity in plasma from various complement deficient mouse strainstested either under lectin activation pathway specific assay conditions,or under classical activation pathway specific assay conditions, asdescribed in Example 22;

FIG. 29B graphically illustrates the time-resolved kinetics of C3convertase activity in plasma from various complement deficient mousestrains tested under lectin activation pathway specific conditions, asdescribed in Example 22;

FIG. 30 illustrates the results of a Western blot analysis showingactivation of human C3, shown by the presence of the a′ chain, bythrombin substrates FXIa and FXa, as described in Example 23;

FIG. 31 shows the results of the C3 deposition assay on serum samplesobtained from WT, MASP-2 (−/−), F11(−/−), F11(−/−)/C4 (−/−) and C4(−/−), as described in Example 23;

FIG. 32A is a Kaplain-Meier survival plot showing the percent survivalover time after exposure to 7.0 Gy radiation in control mice and in micetreated with anti-murine MASP-2 antibody (mAbM11) or anti-human MASP-2antibody (mAbH6) as described in Example 29;

FIG. 32B is a Kaplain-Meier survival plot showing the percent survivalover time after exposure to 6.5 Gy radiation in control mice and in micetreated with anti-murine MASP-2 antibody (mAbM11) or anti-human MASP-2antibody (mAbH6), as described in Example 29;

FIG. 33 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of MASP-2 KO and WT mice after administration of an infectivedose of 2.6×10⁷ cfu of N. meningitidis serogroup A Z2491, demonstratingthat MASP-2 deficient mice are protected from N. meningitidis inducedmortality, as described in Example 30;

FIG. 34 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of MASP-2 KO and WT mice after administration of an infectivedose of 6×10⁶ cfu of N. meningitidis serogroup B strain MC58,demonstrating that MASP-2-deficient mice are protected from N.meningitidis serogroup B strain MC58 induced mortality, as described inExample 30;

FIG. 35 graphically illustrates the log cfu/ml of N. meningitidisserogroup B strain MC58 recovered at different time points in bloodsamples taken from the MASP-2 KO and WT mice after i.p. infection with6×10⁶ cfu of N. meningitidis serogroup B strain MC58 (n=3 at differenttime points for both groups of mice, results are expressed as Means±SEM)demonstrating that although the MASP-2 KO mice were infected with thesame dose of N. meningitidis serogroup B strain MC58 as the WT mice, theMASP-2 KO mice have enhanced clearance of bacteraemia as compared to WT,as described in Example 30;

FIG. 36 graphically illustrates the average illness score of MASP-2 andWT mice at 3, 6, 12 and 24 hours after infection with 6×10⁶ cfu/100 μlN. meningitidis Serogroup Serogroup B strain MC58, demonstrating thatthe MASP-2 deficient mice showed high resistance to the infection, withmuch lower illness scores at 6 hours, as described in Example 30;

FIG. 37 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of mice after administration of an infective dose of 4×10⁶/100μl cfu N. meningitidis Serogroup B strain MC58, followed byadministration 3 hours post infection of either inhibitory anti-MASP-2antibody (1 mg/kg) or control isotype antibody, demonstrating thatanti-MASP-2 antibody is effective to treat and improve survival insubjects infected with N. meningitidis, as described in Example 31;

FIG. 38 graphically illustrates the log cfu/ml of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in 20%human serum concentration after i.p. infection with 6.5×10⁶ cfu/100 μlN. meningitidis serogroup B strain MC58 at 0, 30, 60 and 90 minutesafter incubation in the presence of: (A) normal human serum (NHS) plushuman anti-MASP-2 antibody; (B) normal human serum (NHS) plus isotypecontrol antibody; (C) MBL−/− human serum; (D) normal human serum (NHS)and (E) heat inactivated normal human serum (NHS), showing thatcomplement dependent killing of N. meningitidis in human serum wassignificantly enhanced by the addition of the human anti-MASP-2antibody, as described in Example 32;

FIG. 39 graphically illustrates the log cfu/ml of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in themouse sera samples, demonstrating MASP-2 −/− mouse sera has a higherlevel of bactericidal activity for N. meningitidis than WT mouse sera,as described in Example 32;

FIG. 40 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed mouse erythrocytes (Crry/C3−/−) into the supernatantmeasured by photometry) of mannan-coated murine erythrocytes by humanserum over a range of serum concentrations The sera tested includedheat-inactivated (HI) NHS, MBL−/−, NHS +anti-MASP-2 antibody and NHScontrol, as described in Example 33;

FIG. 41 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed WT mouse erythrocytes into the supernatant measured byphotometry) of non-coated murine erythrocytes by human serum over arange of serum concentrations. The sera tested included heat-inactivated(HI) NHS, MBL−/−, NHS +anti-MASP-2 antibody and NHS control,demonstrating that inhibiting MASP-2 inhibits complement-mediated lysisof non-sensitized WT mouse erythrocytes, as described in Example 33;

FIG. 42 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed mouse erythrocytes (CD55/59 −/−) into the supernatantmeasured by photometry) of non-coated murine erythrocytes by human serumover a range of serum concentrations. The sera tested includedheat-inactivated (HI) NHS, MBL−/−, NHS +anti-MASP-2 antibody and NHScontrol, as described in Example 33;

FIG. 43 graphically illustrates the percent survival over time (days)after exposure to 8.0 Gy radiation in control mice and in mice treatedwith anti-human MASP-2 antibody (mAbH6), as described in Example 34;

FIG. 44 graphically illustrates the time to onset of microvascularocclusion following LPS injection in MASP-2 −/− and WT mice, showing thepercentage of mice with thrombus formation measured over 60 minutes,demonstrating that thrombus formation is detected after 15 minutes in WTmice, with up to 80% of the WT mice demonatrated thrombus formation at60 minutes; in contrast, none of the MASP-2 −/− mice showed any thrombusformation during the 60 minute period (log rank: p=0.0005), as describedin Example 35;

FIG. 45 graphically illustrates the percent survival of saline treatedcontrol mice (n=5) and anti-MASP-2 antibody treated mice (n=5) in theSTX/LPS-induced model of HUS over time (hours), demonstrating that allof the control mice died by 42 hours, whereas, in contrast, 100% of theanti-MASP-2 antibody-treated mice survived throughout the time course ofthe experiment, as described in Example 36;

FIG. 46 graphically illustrates, as a function of time after injuryinduction, the percentage of mice with microvascular occlusion in theFITC/Dextran UV model after treatment with isotype control, or humanMASP-2 antibody mAbH6 (10 mg/kg) dosed at 16 hours and 1 hour prior toinjection of FITC/Dextran, as described in Example 37;

FIG. 47 graphically illustrates the occlusion time in minutes for micetreated with the human MASP-2 antibody (mAbH6) and the isotype controlantibody, wherein the data are reported as scatter-dots with mean values(horizontal bars) and standard error bars (vertical bars). Thestatistical test used for analysis was the unpaired t test; wherein thesymbol “*” indicates p=0.0129, as described in Example 37; and

FIG. 48 graphically illustrates the time until occlusion in minutes forwild-type mice, MASP-2 KO mice, and wild-type mice pre-treated withhuman MASP-2 antibody (mAbH6) administered i.p. at 10 mg/kg 16 hoursbefore, and again 1 hour prior to the induction of thrombosis in theFITC-dextran/light induced endothelial cell injury model of thrombosiswith low light intensity (800-1500), as described in Example 37;

FIG. 49 is a Kaplan-Meier plot showing the percentage of mice withthrombi as a function of time in FITC-Dextran induced thromboticmicroangiopathy in mice treated with increasing doses of human MASP-2inhibitory antibody (mAbH6) or an isotype control antibody, as describedin Example 39;

FIG. 50 graphically illustrates the median time to onset (minutes) ofthrombus formation as a function of mAbH6 dose (*p<0.01 compared tocontrol), as described in Example 39;

FIG. 51 is a Kaplan-Meier plot showing the percentage of mice withmicrovascular occlusion as a function of time in FITC-Dextran inducedthrombotic microangiopathy in mice treated with increasing doses ofhuman MASP-2 inhibitory antibody (mAbH6) or an isotype control antibody,as described in Example 39;

FIG. 52 graphically illustrates the median time to microvascularocclusion as a function of mAbH6 dose (*p<0.05 compared to control), asdescribed in Example 39;

FIG. 53A graphically illustrates the level of MAC deposition in thepresence or absence of human MASP-2 monoclonal antibody (OMS646) underlectin pathway-specific assay conditions, demonstrating that OMS646inhibits lectin-mediated MAC deposition with an IC₅₀ value ofapproximately 1 nM, as described in Example 40;

FIG. 53B graphically illustrates the level of MAC deposition in thepresence or absence of human MASP-2 monoclonal antibody (OMS646) underclassical pathway-specific assay conditions, demonstrating that OMS646does not inhibit classical pathway-mediated MAC deposition, as describedin Example 40;

FIG. 53C graphically illustrates the level of MAC deposition in thepresence or absence of human MASP-2 monoclonal antibody (OMS646) underalternative pathway-specific assay conditions, demonstrating that OMS646does not inhibit alternative pathway-mediated MAC deposition, asdescribed in Example 40;

FIG. 54 graphically illustrates the pharmacokinetic (PK) profile ofhuman MASP-2 monoclonal antibody (OMS646) in mice, showing the OMS646concentration (mean of n=3 animals/groups) as a function of time afteradministration at the indicated dose, as described in Example 40;

FIG. 55A graphically illustrates the pharmacodynamic (PD) response ofhuman MASP-2 monoclonal antibody (OMS646), measured as a drop insystemic lectin pathway activity in mice following intravenousadministration, as described in Example 40;

FIG. 55B graphically illustrates the pharmacodynamic (PD) response ofhuman MASP-2 monoclonal antibody (OMS646), measured as a drop insystemic lectin pathway activity in mice following subcutaneousadministration, as described in Example 40;

FIG. 56 graphically illustrates the inhibitory effect of MASP-2 antibody(OMS646) as compared to sCRI on aHUS serum-induced C5b-9 deposition onADP-activated HMEC-1 cells, as described in Example 41; and

FIG. 57 graphically illustrates the inhibitory effect of MASP-2 antibody(OMS646) as compared to sCRI on aHUS serum-induced thrombus formation onADP-activated HMEC-1 cells, as described in Example 42.

DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO: 1 human MAp19 cDNASEQ ID NO: 2 human MAp19 protein (with leader)SEQ ID NO: 3 human MAp19 protein (mature) SEQ ID NO: 4 human MASP-2 cDNASEQ ID NO: 5 human MASP-2 protein (with leader)SEQ ID NO: 6 human MASP-2 protein (mature)SEQ ID NO: 7 human MASP-2 gDNA (exons 1-6)ANTIGENS: (IN REFERENCE TO THE MASP-2 MATURE PROTEIN)SEQ ID NO: 8 CUBI sequence (aa 1-121)SEQ ID NO: 9 CUBEGF sequence (aa 1-166)SEQ ID NO: 10 CUBEGFCUBII (aa 1-293)SEQ ID NO: 11 EGF region (aa 122-166)SEQ ID NO: 12 serine protease domain (aa 429-671)SEQ ID NO: 13 serine protease domain inactive(aa 610-625 with Ser618 to Ala mutation)SEQ ID NO: 14 TPLGPKWPEPVFGRL (CUB1 peptide)SEQ ID NO: 15 TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI peptide)SEQ ID NO: 16 TFRSDYSN (MBL binding region core)SEQ ID NO: 17 FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding region)SEQ ID NO: 18 IDECQVAPG (EGF PEPTIDE)SEQ ID NO: 19 ANWILCAGLESGGKDSCRGDSGGALV (serine proteasebinding core)Detailed Description PEPTIDE INHIBITORS:SEQ ID NO: 20 MBL full length cDNA SEQ ID NO: 21 MBL full length proteinSEQ ID NO: 22 OGK-X-GP (consensus binding) SEQ ID NO: 23 OGKLGSEQ ID NO: 24 GLR GLQ GPO GKL GPO GSEQ ID NO: 25 GPO GPO GLR GLQ GPO GKL GPO GPO GPOSEQ ID NO: 26 GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOGSEQ ID NO: 27 GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-ficolin)SEQ ID NO: 28 GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPNGAOGEO (human ficolin p35)SEQ ID NO: 29 LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site)EXPRESSION INHIBITORS:SEQ ID NO: 30 cDNA of CUBI-EGF domain (nucleotides 22-680of SEQ ID NO: 4) SEQ ID NO: 31 5′ CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3′Nucleotides 12-45 of SEQ ID NO: 4 including the MASP-2translation start site (sense) SEQ ID NO: 325′GACATTACCTTCCGCTCCGACTCCAACGAGAAG3′Nucleotides 361-396 of SEQ ID NO: 4 encoding a regioncomprising the MASP-2 MBL binding site (sense) SEQ ID NO: 335′AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3′Nucleotides 610-642 of SEQ ID NO: 4 encoding a regioncomprising the CUBII domain CLONING PRIMERS:SEQ ID NO: 34 CGGGATCCATGAGGCTGCTGACCCTC (5′ PCR for CUB)SEQ ID NO: 35 GGAATTCCTAGGCTGCATA (3′ PCR FOR CUB)SEQ ID NO: 36 GGAATTCCTACAGGGCGCT (3′ PCR FOR CUBIEGF)SEQ ID NO: 37 GGAATTCCTAGTAGTGGAT (3′ PCR FOR CUBIEGFCUBII)SEQ ID NOS: 38-47 are cloning primers for humanized antibodySEQ ID NO: 48 is 9 aa peptide bond EXPRESSION VECTOR:SEQ ID NO: 49 is the MASP-2 minigene insertSEQ ID NO: 50 is the murine MASP-2 cDNASEQ ID NO: 51 is the murine MASP-2 protein (w/leader)SEQ ID NO: 52 is the mature murine MASP-2 proteinSEQ ID NO: 53 the rat MASP-2 cDNASEQ ID NO: 54 is the rat MASP-2 protein (w/leader)SEQ ID NO: 55 is the mature rat MASP-2 proteinSEQ ID NO: 56-59 are the oligonucleotides for site-directedmutagenesis of human MASP-2 used to generate human MASP-2ASEQ ID NO: 60-63 are the oligonucleotides for site-directedmutagenesis of murine MASP-2 used to generate murine MASP-2ASEQ ID NO: 64-65 are the oligonucleotides for site-directedmutagenesis of rat MASP-2 used to generate rat MASP-2ASEQ ID NO: 66 DNA encoding 17D20_dc35VH21N11VL (OM5646) heavychain variable region (VH) (without signal peptide)SEQ ID NO: 67 17D20_dc35VH21N11VL (OM5646) heavy chain variableregion (VH) polypeptideSEQ ID NO: 68 17N16mc heavy chain variable region (VH) polypeptideSEQ ID NO: 69: DNA encoding 17D20_dc35VH21N11VL (OM5646) lightchain variable region (VL)SEQ ID NO: 70: 17D20_dc35VH21N11VL (OM5646) light chain variableregion (VL) polypeptideSEQ ID NO: 71: 17N16_dc17N9 light chain variable region (VL) polypeptide

DETAILED DESCRIPTION

The present invention is based upon the surprising discovery by thepresent inventors that it is possible to inhibit the lectin mediatedMASP-2 pathway while leaving the classical pathway intact. The presentinvention also describes the use of MASP-2 as a therapeutic target forinhibiting cellular injury associated with lectin-mediated complementpathway activation while leaving the classical (Clq-dependent) pathwaycomponent of the immune system intact.

I. DEFINITIONS

Unless specifically defined herein, all terms used herein have the samemeaning as would be understood by those of ordinary skill in the art ofthe present invention. The following definitions are provided in orderto provide clarity with respect to the terms as they are used in thespecification and claims to describe the present invention.

As used herein, the term “MASP-2-dependent complement activation”comprises MASP-2-dependent activation of the lectin pathway, whichoccurs under physiological conditions (i.e., in the presence of Ca⁺⁺)leading to the formation of the lectin pathway C3 convertase C4b2a andupon accumulation of the C3 cleavage product C3b subsequently to the C5convertase C4b2a(C3b)n, which has been determined to primarily causeopsonization.

As used herein, the term “alternative pathway” refers to complementactivation that is triggered, for example, by zymosan from fungal andyeast cell walls, lipopolysaccharide (LPS) from Gram negative outermembranes, and rabbit erythrocytes, as well as from many purepolysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumorcells, parasites and damaged cells, and which has traditionally beenthought to arise from spontaneous proteolytic generation of C3b fromcomplement factor C3.

As used herein, the term “lectin pathway” refers to complementactivation that occurs via the specific binding of serum and non-serumcarbohydrate-binding proteins including mannan-binding lectin (MBL),CL-11 and the ficolins (H-ficolin, M-ficolin, or L-ficolin).

As used herein, the term “classical pathway” refers to complementactivation that is triggered by antibody bound to a foreign particle andrequires binding of the recognition molecule C1q.

As used herein, the term “MASP-2 inhibitory agent” refers to any agentthat binds to or directly interacts with MASP-2 and effectively inhibitsMASP-2-dependent complement activation, including anti-MASP-2 antibodiesand MASP-2 binding fragments thereof, natural and synthetic peptides,small molecules, soluble MASP-2 receptors, expression inhibitors andisolated natural inhibitors, and also encompasses peptides that competewith MASP-2 for binding to another recognition molecule (e.g., MBL,H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, but does notencompass antibodies that bind to such other recognition molecules.MASP-2 inhibitory agents useful in the method of the invention mayreduce MASP-2-dependent complement activation by greater than 20%, suchas greater than 50%/a, such as greater than 90%. In one embodiment, theMASP-2 inhibitory agent reduces MASP-2-dependent complement activationby greater than 90% (i.e., resulting in MASP-2 complement activation ofonly 100/% or less).

As used herein, the term “antibody” encompasses antibodies and antibodyfragments thereof, derived from any antibody-producing mammal (e.g.,mouse, rat, rabbit, and primate including human), or from a hybridoma,phage selection, recombinant expression or transgenic animals (or othermethods of producing antibodies or antibody fragments”), thatspecifically bind to a target polypeptide, such as, for example, MASP-2,polypeptides or portions thereof. It is not intended that the term“antibody” limited as regards to the source of the antibody or themanner in which it is made (e.g., by hybridoma, phage selection,recombinant expression, transgenic animal, peptide synthesis, etc).Exemplary antibodies include polyclonal, monoclonal and recombinantantibodies; pan-specific, multispecific antibodies (e.g., bispecificantibodies, trispecific antibodies); humanized antibodies; murineantibodies; chimeric, mouse-human, mouse-primate, primate-humanmonoclonal antibodies; and anti-idiotype antibodies, and may be anyintact antibody or fragment thereof. As used herein, the term “antibody”encompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as dAb, Fab, Fab′, F(ab′)₂, Fv), singlechain (ScFv), synthetic variants thereof, naturally occurring variants,fusion proteins comprising an antibody portion with an antigen-bindingfragment of the required specificity, humanized antibodies, chimericantibodies, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen-binding site or fragment (epitoperecognition site) of the required specificity.

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an epitope. Monoclonal antibodies are highlyspecific for the target antigen. The term “monoclonal antibody”encompasses not only intact monoclonal antibodies and full-lengthmonoclonal antibodies, but also fragments thereof (such as Fab, Fab′,F(ab′)₂, Fv), single chain (ScFv), variants thereof, fusion proteinscomprising an antigen-binding portion, humanized monoclonal antibodies,chimeric monoclonal antibodies, and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen-binding fragment(epitope recognition site) of the required specificity and the abilityto bind to an epitope. It is not intended to be limited as regards thesource of the antibody or the manner in which it is made (e.g., byhybridoma, phage selection, recombinant expression, transgenic animals,etc.). The term includes whole immunoglobulins as well as the fragmentsetc. described above under the definition of “antibody”.

As used herein, the term “antibody fragment” refers to a portion derivedfrom or related to a full-length antibody, such as, for example, ananti-MASP-2 antibody, generally including the antigen binding orvariable region thereof. Illustrative examples of antibody fragmentsinclude Fab, Fab′, F(ab)₂, F(ab′)₂ and Fv fragments, scFv fragments,diabodies, linear antibodies, single-chain antibody molecules andmultispecific antibodies formed from antibody fragments.

As used herein, a “single-chain Fv” or “scFv” antibody fragmentcomprises the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains, which enables the scFv to form the desired structure forantigen binding.

As used herein, a “chimeric antibody” is a recombinant protein thatcontains the variable domains and complementarity-determining regionsderived from a non-human species (e.g., rodent) antibody, while theremainder of the antibody molecule is derived from a human antibody.

As used herein, a “humanized antibody” is a chimeric antibody thatcomprises a minimal sequence that conforms to specificcomplementarity-determining regions derived from non-humanimmunoglobulin that is transplanted into a human antibody framework.Humanized antibodies are typically recombinant proteins in which onlythe antibody complementarity-determining regions are of non-humanorigin.

As used herein, the term “mannan-binding lectin” (“MBL”) is equivalentto mannan-binding protein (“MBP”).

As used herein, the “membrane attack complex” (“MAC”) refers to acomplex of the terminal five complement components (C5b combined withC6, C7, C8 and C-9) that inserts into and disrupts membranes (alsoreferred to as C5b-9).

As used herein, “a subject” includes all mammals, including withoutlimitation humans, non-human primates, dogs, cats, horses, sheep, goats,cows, rabbits, pigs and rodents.

As used herein, the amino acid residues are abbreviated as follows:alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine(Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q),glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L),lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline(Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine(Tyr;Y), and valine (Val;V).

In the broadest sense, the naturally occurring amino acids can bedivided into groups based upon the chemical characteristic of the sidechain of the respective amino acids. By “hydrophobic” amino acid ismeant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By“hydrophilic” amino acid is meant either Gly, Asn, Gin, Ser, Thr, Asp,Glu, Lys, Arg or His. This grouping of amino acids can be furthersubclassed as follows. By “uncharged hydrophilic” amino acid is meanteither Ser, Thr, Asn or Gin. By “acidic” amino acid is meant either Gluor Asp. By “basic” amino acid is meant either Lys, Arg or His.

As used herein the term “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine.

The term “oligonucleotide” as used herein refers to an oligomer orpolymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) ormimetics thereof. This term also covers those oligonucleobases composedof naturally-occurring nucleotides, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring modifications.

As used herein, an “epitope” refers to the site on a protein (e.g., ahuman MASP-2 protein) that is bound by an antibody. “Overlappingepitopes” include at least one (e.g., two, three, four, five, or six)common amino acid residue(s), including linear and non-linear epitopes.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably and mean any peptide-linked chain of amino acids,regardless of length or post-translational modification. The MASP-2protein described herein can contain or be wild-type proteins or can bevariants that have not more than 50 (e.g., not more than one, two,three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35,40, or 50) conservative amino acid substitutions. Conservativesubstitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine, and leucine; asparticacid and glutamic acid; asparagine, glutamine, serine and threonine;lysine, histidine and arginine; and phenylalanine and tyrosine.

In some embodiments, the human MASP-2 protein can have an amino acidsequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100) % identical to the human MASP-2 proteinhaving the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, peptide fragments can be at least 6 (e.g., at least7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, or 600 or more) amino acid residues in length (e.g., at least6 contiguous amino acid residues of SEQ ID NO: 5). In some embodiments,an antigenic peptide fragment of a human MASP-2 protein is fewer than500 (e.g., fewer than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190,180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65,60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues in length(e.g., fewer than 500 contiguous amino acid residues in any one of SEQID NOS: 5).

Percent (%) amino acid sequence identity is defined as the percentage ofamino acids in a candidate sequence that are identical to the aminoacids in a reference sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Alignment for purposes of determining percent sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared can be determined by known methods.

II. OVERVIEW OF THE INVENTION

Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-11) are thespecific recognition molecules that trigger the innate complement systemand the system includes the lectin initiation pathway and the associatedterminal pathway amplification loop that amplifies lectin-initiatedactivation of terminal complement effector molecules. C1q is thespecific recognition molecule that triggers the acquired complementsystem and the system includes the classical initiation pathway andassociated terminal pathway amplification loop that amplifiesC1q-initiated activation of terminal complement effector molecules. Werefer to these two major complement activation systems as thelectin-dependent complement system and the C1q-dependent complementsystem, respectively.

In addition to its essential role in immune defense, the complementsystem contributes to tissue damage in many clinical conditions. Thus,there is a pressing need to develop therapeutically effective complementinhibitors to prevent these adverse effects. With the recognition thatit is possible to inhibit the lectin mediated MASP-2 pathway whileleaving the classical pathway intact comes the realization that it wouldbe highly desirable to specifically inhibit only the complementactivation system causing a particular pathology without completelyshutting down the immune defense capabilities of complement. Forexample, in disease states in which complement activation is mediatedpredominantly by the lectin-dependent complement system, it would beadvantageous to specifically inhibit only this system. This would leavethe Clq-dependent complement activation system intact to handle immunecomplex processing and to aid in host defense against infection.

The preferred protein component to target in the development oftherapeutic agents to specifically inhibit the lectin-dependentcomplement system is MASP-2. Of all the known protein components of thelectin-dependent complement system (MBL, H-ficolin, M-ficolin,L-ficolin, MASP-2, C2-C9, Factor B, Factor D, and properdin), onlyMASP-2 is both unique to the lectin-dependent complement system andrequired for the system to function. The lectins (MBL, H-ficolin,M-ficolin, L-ficolin and CL-11) are also unique components in thelectin-dependent complement system. However, loss of any one of thelectin components would not necessarily inhibit activation of the systemdue to lectin redundancy. It would be necessary to inhibit all fivelectins in order to guarantee inhibition of the lectin-dependentcomplement activation system. Furthermore, since MBL and the ficolinsare also known to have opsonic activity independent of complement,inhibition of lectin function would result in the loss of thisbeneficial host defense mechanism against infection. In contrast, thiscomplement-independent lectin opsonic activity would remain intact ifMASP-2 was the inhibitory target. An added benefit of MASP-2 as thetherapeutic target to inhibit the lectin-dependent complement activationsystem is that the plasma concentration of MASP-2 is among the lowest ofany complement protein (=500 ng/ml); therefore, correspondingly lowconcentrations of high-affinity inhibitors of MASP-2 may be sufficientto obtain full inhibition (Moller-Kristensen, M., et al., J. ImmunolMethods 282:159-167, 2003).

III. THE ROLE OF MASP-2 IN THROMBOTIC MICROANGIOPATHIES AND THERAPEUTICMETHODS USING MASP-2 INHIBITORY AGENTS

Overview

Thrombotic microangiopathy (TMA) is a pathology characterized by bloodclots in small blood vessels (Benz, K.; et al., Curr Opin NephrolHypertens 19(3):242-7 (2010)). Stress or injury to the underlyingvascular endothelium is believed to be a primary driver. Clinical andlaboratory findings of TMA include thrombocytopenia, anemia, purpura,and renal failure. The classic TMAs are hemolytic uremic syndrome (HUS)and thrombotic thrombocytopenic purpura (TTP). The characteristicunderlying pathological feature of TMAs are platelet activation and theformation of microthrombi in the small arterioles and venules.Complement activation initiated, at least in part, by an injury orstress to microvascular endothelium, is also implicated in other TMAsincluding catastrophic antiphospholipid syndrome (CAPS), systemic Degosdisease, and TMAs secondary to cancer, cancer chemotherapy andtransplantation.

Direct evidence for a pathological role of complement in a host ofnephritides is provided by studies of patients with genetic deficienciesin specific complement components. A number of reports have documentedan association of renal injury with deficiencies of complementregulatory factor H (Ault, B. H., Nephrol. 14:1045-1053, 2000; Levy, M.,et al., Kidney Int. 30:949-56, 1986; Pickering, M. C., et al., Nat.Genet. 31:424-8, 2002). Factor H deficiency results in low plasma levelsof factor B and C3 due to activation-related consumption of thesecomponents. Circulating levels of C5b-9 are also elevated in the serumof these patients, implying complement activation. Membranoproliferativeglomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS)are associated with factor H deficiency or mutations of factor H. FactorH-deficient pigs (Jansen, J. H., et al., Kidney Int. 53:331-49, 1998)and factor-H knockout mice (Pickering, M. C., 2002) display MPGN-likesymptoms, confirming the importance of factor H in complementregulation. Deficiencies of other complement components are associatedwith renal disease, secondary to the development of systemic lupuserythematosus (SLE) (Walport, M. J., Davies, et al., Ann. N.Y. Acad.Sci. 815:267-81, 1997). Deficiency for C1q, C4 and C2 predisposestrongly to the development of SLE via mechanisms relating to defectiveclearance of immune complexes and apoptotic material. In many of theseSLE patients lupus nephritis occurs, characterized by the deposition ofimmune complexes throughout the glomerulus.

aHUS

Atypical hemolytic uremic syndrome (aHUS) is part of a group ofconditions termed “Thrombotic microangiopathies.” In the atypical formof HUS (aHUS), the disease is associated with defective complementregulation and can be either sporadic or familial. Familial cases ofaHUS are associated with mutations in genes coding for complementactivation or complement regulatory proteins, including complementfactor H, factor I, factor B, membrane cofactor CD46 as well ascomplement factor H-related protein 1 (CFHR1) and complement factorH-related protein 3 (CFHR3). (Zipfel, P. F., et al., PloS Genetics3(3):e41 (2007)). The unifying feature of this diverse array of geneticmutations associated with aHUS is a predisposition to enhancedcomplement activation on cellular or tissue surfaces. Therefore, oneaspect of the present invention comprises treating a patient sufferingwith aHUS that is associated with a factor H defiency by administeringan effective amount of a MASP-2 inhibitory agent. Another aspect of thepresent invention comprises treating a patient suffering with HUS thatis associated with a factor I, factor B, membrane cofactor CD46, CFHR1or CFHR3 deficiency by administering an effective amount of a MASP-2inhibitory agent.

Significant progress has been made recently toward the understanding ofthe molecular pathophysiology underlying enhanced complement activationin aHUS caused by the diverse set of mutant complement factors. Thismechanism is best understood for factor H mutations. Factor H is anabundant serum protein comprising 20 short consensus repeat (SCR)domains that acts as a negative regulator of complement activation bothin solution as well as on host cell surfaces. It targets the activatedform of C3 and, together with factor I and other cofactors, promotes itsinactivation, forestalling further complement activation. To effectivelycontrol complement activation on host cell surfaces, factor H needs tointeract with host cells, which is mediated by SCR domains 16-20. Allfactor H mutations associated with aHUS described to date are clusteredin the C-terminal region encompassing (SCR) domains 16-20. These mutantfactor H proteins are fully functional in controlling C3 activation insolution, but are unable to interact with host cell surfaces andconsequently cannot control C3 activation on cellular surfaces (Exp Med204(6):1249-56 (2007)). Thus, certain mutations of factor H areassociated with aHUS because the mutant factor H protein fails tointeract with host cell surfaces and thus cannot effectively downmodulate complement activation on host cell surfaces, including themicrovascular endothelium. As a result, once initial C3 activation hasoccurred, subsequent complement activation on microvascular endothelialsurfaces proceeds unabated in patients with factor H mutations. Thisuncontrolled activation of complement ultimately leads to progressiveinjury to the vascular endothelium, subsequent platelet aggregation andmicrovascular coagulation, and hemolysis caused by sheer stress of RBCpassage through partially occluded microvessels. Thus, aHUS diseasemanifestations and clinical and laboratory findings are directly linkedto a defect in the negative regulation of complement on the surface ofthe microvascular endothelium.

Analogous to factor H mutation, loss-of-function mutations in thenegative complement modulators factor I and membrane cofactor protein(CD46) are also linked to aHUS. The opposite has been observed forfactor B the C3 protein in that aHUS was found to be associated withgain-of-function mutations in these proteins (Pediatr Nephrol25(12):2431-42 (2010)). Thus, a host of converging data implicatescomplement activation in aHUS pathogenesis. This notion is mostconvincingly supported by the therapeutic efficacy ofeculizumab, amonoclonal antibody that blocks the terminal complement protein C5 inthe treatment of aHUS.

While the central role of complement as an effector mechanism in aHUS iswidely accepted, the triggers initiating complement activation and themolecular pathways involved are unresolved. Not all individuals carryingthe above described mutations develop aHUS. In fact, familial studieshave suggested that the penetrance of aHUS is only ˜50% (Ann Hum Genet74(1): 17-26 (2010)). The natural history of the disease suggests thataHUS most often develops after an initiating event such as an infectiousepisode or an injury. Infectious agents are well known to activate thecomplement system. In the absence of pre-existing adaptive immunity,complement activation by infectious agents may be primarily initiatedvia the lectin pathway. Thus, lectin pathway activation triggered by aninfection may represent the initiating trigger for subsequentpathological amplification of complement activation in aHUS-predisposedindividuals, which may ultimately lead to disease progression.Accordingly, another aspect of the present invention comprises treatinga patient suffering with aHUS secondary to an infection by administeringan effective amount of a MASP-2 inhibitory agent.

Other forms of injury to host tissue will activate complement via thelectin pathway, in particular injury to the vascular endothelium. Humanvascular endothelial cells subject to oxidative stress for examplerespond by expressing surface moieties that bind lectins and activatethe lectin pathway of complement (Am J. Pathol 156(6): 1549-56 (2000)).Vascular injury following ischemia/reperfusion also activates complementvia the lectin pathway in vivo (Scand J Immunol 61(5):426-34 (2005)).Lectin pathway activation in this setting has pathological consequencesfor the host, and inhibition of the lectin pathway by blocking MASP-2prevents further host tissue injury and adverse outcomes (Schwaeble PNAS2011).

Thus, other processes that precipitate aHUS are also known to activatethe lectin pathway of complement. It is therefore likely that the lectinpathway may represent the initial complement activating mechanism thatis inappropriately amplified in a deregulated fashion in individualsgenetically predisposed to aHUS, thus initiating aHUS pathogenesis. Byinference, agents that block activation of complement via the lectinpathway, including anti-MASP-2 antibodies, are expected to preventdisease progression or reduce exacerbations in aHUS susceptibleindividuals.

In further support of this concept, recent studies have identified S.pneumonia as an important etiological agent in pediatric cases of aHUS.(Nephrology (Carlton), 17:48-52 (2012); Pediatr Infect Dis J.30(9):736-9 (2011)). This particular etiology appears to have anunfavorable prognosis, with significant mortality and long-termmorbidity. Notably, these cases involved non-enteric infections leadingto manifestations of microangiopathy, uremia and hemolysis withoutevidence of concurrent mutations in complement genes known to predisposeto aHUS. It is important to note that S. pneumonia is particularlyeffective at activating complement, and does so predominantly throughthe lectin pathway. Thus, in cases of non-enteric HUS associated withpneumococcal infection, manifestations of microangiopathy, uremia andhemolysis are expected to be driven predominantly by activation of thelectin pathway, and agents that block the lectin pathway, includinganti-MASP-2 antibodies, are expected to prevent progression of aHUS orreduce disease severity in these patients. Accordingly, another aspectof the present invention comprises treating a patient suffering withnon-enteric aHUS that is associated with S. pneumonia infection byadministering an effective amount of a MASP-2 inhibitory agent.

In accordance with the foregoing, in some embodiments, in the setting ofa subject at risk for developing renal failure associated with aHUS, amethod is provided for decreasing the likelihood of developing aHUS, orof developing renal failure associated with aHUS, comprisingadministering an amount of an MASP-2 inhibitory agent for a time periodeffective to ameliorate or prevent renal failure in the subject. In someembodiments, the method further comprises the step of determiningwhether a subject is at risk for developing aHUS prior to the onset ofany symptoms associated with aHUS. In other embodiments, the methodcomprises determining whether a subject is a risk for developing aHUSupon the onset of at least one or more symptoms indicative of aHUS(e.g., the presence of anemia, thrombocytopenia and/or renalinsufficiency) and/or the presence of thrombotic microangiopathy in abiopsy obtained from the subject. The determination of whether a subjectis at risk for developing aHUS comprises determining whether the subjecthas a genetic predisposition to developing aHUS, which may be carriedout by assessing genetic information (e.g. from a database containingthe genotype of the subject), or performing at least one geneticscreening test on the subject to determine the presence or absence of agenetic marker associated with aHUS (i.e., determining the presence orabsence of a genetic mutation associated with aHUS in the genes encodingcomplement factor H (CFH), factor I (CFI), factor B (CFB), membranecofactor CD46, C3, complement factor H-related protein 1 (CFHR1), orTHBD (encoding the anticoagulant protein thrombodulin) or complementfactor H-related protein 3 (CFHR3), or complement factor H-relatedprotein 4 (CFHR4)) either via genome sequencing or gene-specificanalysis (e.g., PCR analysis), and/or determining whether the subjecthas a family history of aHUS. Methods of genetic screening for thepresence or absence of a genetic mutation associated with aHUS are wellestablished, for example, see Noris M et al. “Atypical Hemolytic-UremicSyndrome,” 2007 November 16 [Updated 2011 Mar. 10]. In: Pagon R A, BirdT D, Dolan C R, et al., editors. GeneReviews™, Seattle (Wash.):University of Washington, Seattle.

For example, overall the penetrance of the disease in those withmutations of complement factor H (CFH) is 48%, and the penetrance formutations in CD46 is 53%, for mutations in CFI is 50%, for mutations inC3 is 56% and for mutations in THBD is 64% (Caprioli J. et al., Blood,108:1267-79 (2006); Noris et al., Clin J Am Soc Nephrol 5:1844-59(2010)). As described in Caprioli et al., (2006), supra, a substantialnumber of individuals with a mutation in complement Factor H (CFH) neverdevelop aHUS, and it is postulated that suboptimal CFH activity in theseindividuals is sufficient to protect the host from the effects ofcomplement activation in physiological conditions, however, suboptimalCFH activity is not sufficient to prevent C3b from being deposited onvascular endothelial cells when exposure to an agent that activatescomplement produces higher than normal amounts of C3b.

Accordingly, in one embodiment, a method is provided for inhibitingMASP-2-dependent complement activation in a subject suffering from, orat risk for developing non-Factor H-dependent atypical hemolytic uremicsyndrome, comprising administering to the subject a compositioncomprising an amount of a MASP-2 inhibitory agent effective to inhibitMASP-2-dependent complement activation. In another embodiment, a methodis provided for inhibiting MASP-2-dependent complement activation in asubject at risk for developing Factor H-dependent atypical hemolyticuremic syndrome, comprising periodically monitoring the subject todetermine the presence or absence of anemia, thrombocytopenia or risingcreatinine, and treating with a MASP-2 inhibitory agent upon thedetermination of the presence of anemia thrombocytopenia, or risingcreatinine. In another embodiment, a method is provided for reducing thelikelihood that a subject at risk for developing Factor H-dependent aHUSwill suffer clinical symptoms associated with aHUS, comprisingadministering a MASP-2 inhibitory agent prior to, or during, or after anevent known to be associated with triggering aHUS clinical symptoms, forexample, drug exposure (e.g., chemotherapy), infection (e.g., bacterialinfection), malignancy, an injury, organ or tissue transplant, orpregnancy.

In one embodiment, a method is provided for reducing the likelihood thata subject at risk for developing aHUS will suffer clinical symptomsassociated with aHUS, comprising periodically monitoring the subject todetermine the presence or absence of anemia, thrombocytopenia or risingcreatinine, and treating with a MASP-2 inhibitory agent upon thedetermination of the presence of anemia, thrombocytopenia, or risingcreatinine.

In another embodiment, a method is provided for reducing the likelihoodthat a subject at risk for developing aHUS will suffer clinical symptomsassociated with aHUS comprising administering a MASP-2 inhibitory agentprior to, or during, or after an event known to be associated withtriggering aHUS clinical symptoms, for example, drug exposure (e.g.,chemotherapy), infection (e.g., bacterial infection), malignancy, aninjury, organ or tissue transplant, or pregnancy.

In some embodiments, the MASP-2 inhibitory agent is administered for atime period of at least one, two, three, four days, or longer, prior to,during, or after the event associated with triggering aHUS clinicalsymptoms and may be repeated as determined by a physician until thecondition has been resolved or is controlled. In a pre-aHUS setting, theMASP-2 inhibitory agent may be administered to the subject systemically,such as by intra-arterial, intravenous, intramuscular, inhalational,nasal, subcutaneous or other parenteral administration.

In some embodiments, in the setting of initial diagnosis of aHUS, or ina subject exhibiting one or more symptoms consistent with a diagnosis ofaHUS (e.g., the presence of anemia, thrombocytopenia and/or renalinsufficiency), the subject is treated with an effective amount of aMASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) as a first linetherapy in the absence of plasmapheresis, or in combination withplasmapheresis. As a first line therapy, the MASP-2 inhibitory agent maybe administered to the subject systemically, such as by intra-arterial,intravenous, intramuscular, inhalational, nasal, subcutaneous or otherparenteral administration. In some embodiments, the MASP-2 inhibitoryagent is administered to a subject as a first line therapy in theabsence of plasmaphersis to avoid the potential complications ofplasmaphersis including hemorrhage, infection, and exposure to disordersand/or allergies inherent in the plasma donor, or in a subject otherwiseaverse to plasmapheresis, or in a setting where plasmapheresis isunavailable.

In some embodiments, the method comprises administering a MASP-2inhibitory agent to a subject suffering from aHUS via a catheter (e.g.,intravenously) for a first time period (e.g., at least one day to a weekor two weeks) followed by administering a MASP-2 inhibitory agent to thesubject subcutaneously for a second time period (e.g., a chronic phaseof at least two weeks or longer). In some embodiments, theadministration in the first and/or second time period occurs in theabsence of plasmapheresis. In some embodiments, the method furthercomprises determining the level of at least one complement factor (e.g.,C3, C5) in the subject prior to treatment, and optionally duringtreatment, wherein the determination of a reduced level of at least onecomplement factor in comparison to a standard value or healthy controlsubject is indicative of the need for continued treatment with theMASP-2 inhibitory agent.

In some embodiments, the method comprises administering a MASP-2inhibitory agent, such as an anti-MASP-2 antibody, to a subjectsuffering from, or at risk for developing, aHUS either intravenously,intramuscularly, or preferably, subcutaneously. Treatment may be chronicand administered daily to monthly, but preferably every two weeks. Theanti-MASP-2 antibody may be administered alone, or in combination with aC5 inhibitor, such as eculizamab.

HUS

Like atypical HUS, the typical form of HUS displays all the clinical andlaboratory findings of a TMA. Typical HUS, however, is often a pediatricdisease and usually has no familial component or direct association withmutations in complement genes. The etiology of typical HUS is tightlylinked to infection with certain intestinal pathogens. The patientstypically present with acute renal failure, hemoglobinuria, andthrombocytopenia, which typically follows an episode of bloody diarrhea.The condition is caused by an enteric infection with Shigelladissenteria, Salmonella or shiga toxin-like producing enterohemorrhagicstrains of E. coli. such as E. coli O157:H7. The pathogens are acquiredfrom contaminated food or water supply. HUS is a medical emergency andcarries a 5-10% mortality. A significant portion of survivors developchronic kidney disease (Corrigan and Boineau, Pediatr Rev 22 (11): 365-9(2011)) and may require kidney transplantation.

The microvascular coagulation in typical HUS occurs predominantly,though not exclusively, in the renal microvasculature. The underlyingpathophysiology is mediated by Shiga toxin (STX). Excreted byenteropathic microbes into the intestinal lumen, STX crosses theintestinal barrier, enters the bloodstream and binds to vascularendothelial cells via the blobotriaosyl ceramide receptor CD77 (Boyd andLingwood Nephron 51:207 (1989)), which is preferentially expressed onglomerular endothelium and mediates the toxic effect of STX. Once boundto the endothelium, STX induces a series of events that damage vascularendothelium, activate leukocytes and cause vWF-dependent thrombusformation (Forsyth et al., Lancet 2: 411-414 (1989); Zoja et al., KidneyInt. 62: 846-856 (2002); Zanchi et al., J. Immunol. 181:1460-1469(2008); Morigi et al., Blood 98: 1828-1835 (2001); Guessou et al.,Infect. Immun., 73: 8306-8316 (2005)). These microthrombi obstruct orocclude the arterioles and capillaries of the kidney and other organs.The obstruction of blood flow in arterioles and capillaries bymicrothrombi increases the shear force applied to RBCs as they squeezethrough the narrowed blood vessels. This can result in destruction ofRBC by shear force and the formation of RBC fragments calledschistocytes. The presence of schistocytes is a characteristic findingin HUS. This mechanism is known as microangiopathic hemolysis. Inaddition, obstruction of blood flow results in ischemia, initiating acomplement-mediated inflammatory response that causes additional damageto the affected organ.

The lectin pathway of complement contributes to the pathogenesis of HUSby two principle mechanisms: 1) MASP-2-mediated direct activation of thecoagulation cascade caused by endothelial injury, and 2) lectin-mediatedsubsequent complement activation induced by the ischemia resulting fromthe initial occlusion of microvascular blood flow.

STX injures microvascular endothelial cells, and injured endothelialcells are known to activate the complement system. As detailed above,complement activation following endothelial cell injury is drivenpredominantly by the lectin pathway. Human vascular endothelial cellssubject to oxidative stress respond by expressing surface moieties thatbind lectins and activate the lectin pathway of complement (Collard etal., Am J Pathol. 156(5):1549-56 (2000)). Vascular injury followingischemia reperfusion also activates complement via the lectin pathway invivo (Scand J Immunol 61(5):426-34 (2005)). Lectin pathway activation inthis setting has pathological consequences for the host, and inhibitionof the lectin pathway by blockade of MASP-2 prevents further host tissueinjury and adverse outcomes (Schwaeble et al., PNAS (2011)). In additionto complement activation, lectin-dependent activation of MASP-2 has beenshown to result in cleavage of prothrombin to form thrombin and topromote coagulation. Thus, activation of the lectin pathway ofcomplement by injured endothelial cells can directly activate thecoagulation system. The lectin pathway of complement, by virtue ofMASP-2-mediated prothombin activation, therefore is likely the dominantmolecular pathway linking the initial endothelial injury by STX to thecoagulation and microvascular thrombosis that occurs in HUS. It istherefore expected that lectin pathway inhibitors, including, but notlimited to, antibodies that block MASP-2 function, will prevent ormitigate microvascular coagulation, thrombosis and hemolysis in patientssuffering from HUS. Indeed, administration of anti-MASP-2 antibodyprofoundly protects mice in a model of typical HUS. As described inExample 36 and shown in FIG. 45, all control mice exposed to STX and LPSdeveloped severe HUS and became moribund or died within 48 hours. On theother hand, as further shown in FIG. 45, all mice treated with ananti-MASP-2 antibody and then exposed to STX and LPS survived (Fisher'sexact p<0.01; N=5). Thus, anti-MASP-2 therapy profoundly protects micein this model of HUS. It is expected that administration of a MASP-2inhibitory agent, such as a MASP-2 antibody, will be effective in thetreatment of HUS patients and provide protection from microvascularcoagulation, thrombosis, and hemolysis caused by infection withenteropathic E. coli or other STX-producing pathogens.

While shown here for HUS caused by STX, it is expected that anti-MASP-2therapy will also be beneficial for HUS-like syndromes due toendothelial injury caused by other toxic agents. This includes agentssuch as mitomycin, ticlopidine, cycplatin, quinine, cyclosporine,bleomycin as well as other chemotherapy drugs and immunosuppresssivedrugs. Thus, it is expected that anti-MASP-2 antibody therapy, or othermodalities that inhibit MASP-2 activity, will effectively prevent orlimit coagulation, thrombus formation, and RBC destruction and preventrenal failure in HUS and other TMA related diseases (i.e., aHUS andTTP).

Patients suffering from HUS often present with diarrhea and vomiting,their platelet counts are usually reduced (thrombocytopenia), and RBCsare reduced (anemia). A pre-HUS diarrhea phase typically lasts for aboutfour days, during which subjects at risk for developing HUS typicallyexhibit one or more of the following symptoms in addition to severediarrhea: a hematocrit level below 30% with smear evidence ofintravascular erythrocyte destruction, thrombocytopenia (platelet count<150×10³/mm³), and/or the presence of impaired renal function (serumcreatinine concentration greater than the upper limit of reference rangefor age). The presence of oligoanuria (urine output ≤0.5 mL/kg/h for >1day) can be used as a measure for progression towards developing HUS(see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884-889 (2011)).Testing is typically carried out for the presence of infection with E.coli bacteria (E. coli O157:H7), or Shigella or Salmonella species. In asubject testing positive for infection with enterogenic E. coli (e.g.,E. coli O157:H7), the use of antibiotics is contra-indicated because theuse of antibiotics may increase the risk of developing HUS throughincreased STX production (See Wong C. et al., N Engl J. Med342:1930-1936 (2000). For subjects testing positive for Shigella orSalmonella, antibiotics are typically administered to clear theinfection. Other well established first-line therapy for HUS includesvolume expansion, dialysis and plasmapheresis.

In accordance with the foregoing, in some embodiments, in the setting ofa subject suffering from one or more symptoms associated with a pre-HUSphase and at risk for developing HUS (i.e., the subject exhibits one ormore of the following: diarrhea, a hematocrit level less than 30% withsmear evidence of intravascular erythrocyte destruction,thrombocytopenia (platelet count less than 150×10³/mm³), and/or thepresence of impaired renal function (serum creatinine concentrationgreater than the upper limit of reference range for age)), a method isprovided for decreasing the risk of developing HUS, or of decreasing thelikelihood of renal failure in the subject, comprising administering anamount of an MASP-2 inhibitory agent for a time period effective toameliorate or prevent impaired renal function. In some embodiments, theMASP-2 inhibitory agent is administered for a time period of at leastone, two, three, four or more days, and may be repeated as determined bya physician until the condition has been resolved or is controlled. In apre-HUS setting, the MASP-2 inhibitory agent may be administered to thesubject systemically, such as by intra-arterial, intravenous,intramuscular, inhalational, nasal, oral, subcutaneous or otherparenteral administration.

The treatment of E. coli O157:H7 infection with bactericidalantibiotics, particularly β-lactams, has been associated with anincreased risk of developing HUS (Smith et al., Pediatr Infect Dis J31(1):37-41 (2012). In some embodiments, in the setting of a subjectsuffering from symptoms associated with a pre-HUS phase, wherein thesubject is known to have an infection with enterogenic E. coli for whichthe use of antibiotics is contra-indicated (e.g., E. coli O157:H7), amethod is provided for decreasing the risk of developing HUS, or ofdecreasing the likelihood of renal failure in the subject, comprisingadministering an amount of a MASP-2 inhibitory agent for a first timeperiod effective to inhibit or prevent the presence of oligoanuria inthe subject (e.g., at least one, two, three or four days), wherein theadministration of the MASP-2 inhibitory agent during the first timeperiod occurs in the absence of an antibiotic. In some embodiments, themethod further comprises administering the MASP-2 inhibitory agent tothe subject in combination with an antibiotic for a second time period(such as at least one to two weeks).

In other embodiments, in the setting of a subject suffering fromsymptoms associated with a pre-HUS phase, wherein the subject is knownto have an infection with Shigella or Salmonella, a method is providedfor decreasing the risk of developing HUS, or of decreasing thelikelihood of renal failure in the subject, comprising administering anamount of a MASP-2 inhibitory agent and for a time period effective toinhibit or prevent the presence of oligoanuria in the subject, whereinthe administration of the MASP-2 inhibitory agent is either in thepresence or in the absence of a suitable antibiotic.

In some embodiments, in the setting of an initial diagnosis of HUS, orin a subject exhibiting one or more symptoms consistent with a diagnosisof HUS (e.g., the presence of renal failure, or microangiopathichemolytic anemia in the absence of low fibrinogen, or thrombocytopenia)the subject is treated with an effective amount of a MASP-2 inhibitoryagent (e.g. a anti-MASP-2 antibody) as a first-line therapy in theabsence of plasmapheresis, or in combination with plasmapheresis. As afirst-line therapy, the MASP-2 inhibitory agent may be administered tothe subject systemically, such as by intra-arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration. In some embodiments, the MASP-2 inhibitory agent isadministered to a subject as a first line therapy in the absence ofplasmapheresis to avoid the complications of plasmapheresis such ashemorrhage, infection, and exposure to disorders and/or allergiesinherent in the plasma donor, or in a subject otherwise averse toplasmaphoresis, or in a setting where plasmapheresis is unavailable.

In some embodiments, the method comprises administering a MASP-2inhibitory agent to a subject suffering from HUS via a catheter (e.g.,intravenously) for a first time period (e.g., an acute phase lasting atleast one day to a week or two weeks) followed by administering a MASP-2inhibitory agent to the subject subcutaneously for a second time period(e.g., a chronic phase of at least two weeks or longer). In someembodiments, the administration in the first and/or second time periodoccurs in the absence of plasmapheresis. In some embodiments, the methodfurther comprises determining the level of at least one complementfactor (e.g., C3, C5) in the subject prior to treatment, and optionallyduring treatment, wherein the determination of a reduced level of the atleast one complement factor in comparison to a standard value or healthycontrol subject is indicative of the need for treatment, and wherein thedetermination of a normal level is indicative of improvement.

In some embodiments, the method comprises administering a MASP-2inhibitory agent, such as an anti-MASP-2 antibody, to a subjectsuffering from, or at risk for developing, HUS either subcutaneously orintravenously. Treatment is preferably daily, but can be as infrequentas weekly or monthly. Treatment will continue for at least one week andas long as 3 months. The anti-MASP-2 antibody may be administered alone,or in combination with a C5 inhibitor, such as eculizamab.

TTP:

Thrombotic thrombocytopenic purpura (TTP) is a life threatening disorderof the blood-coagulation system, caused by autoimmune or hereditarydysfunctions that activate the coagulation cascade or the complementsystem (George, J N, N Engl J Med; 354:1927-35 (2006)). This results innumerous microscopic clots, or thomboses, in small blood vesselsthroughout the body. Red blood cells are subjected to shear stress whichdamages their membranes, leading to intravascular hemolysis. Theresulting reduced blood flow and endothelial injury results in organdamage, including brain, heart, and kidneys. TTP is clinicallycharacterized by thrombocytopenia, microangiopathic hemolytic anemia,neurological changes, renal failure and fever. In the era before plasmaexchange, the fatality rate was 90% during acute episodes. Even withplasma exchange, survival at six months is about 80%.

TTP may arise from genetic or acquired inhibition of the enzymeADAMTS-13, a metalloprotease responsible for cleaving large multimers ofvon Willebrand factor (vWF) into smaller units. ADAMTS-13 inhibition ordeficiency ultimately results in increased coagulation (Tsai, H. J AmSoc Nephrol 14: 1072-1081, (2003)). ADAMTS-13 regulates the activity ofvWF; in its absence, vWF forms large multimers which are more likely tobind platelets and predisposes patients to platelet aggregation andthrombosis in the microvasculature.

Upshaw-Schulman syndrome (USS, also described as congenital TTP) is acongenital deficiency of ADAMTS13 activity due to ADAMTS13 genemutations (Schulman et al., Blood, 16(1):943-57, 1960; Upshaw et al.,New Engl. J. Med, 298 (24):1350-2, 1978). Numerous mutations in ADAMTS13have been identified in individuals with congenital TTP (Kinoshita etal., International Journal of Hematology, 74:101-108 (2001); Levy etal., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS99(18):11902-11907 (2002); Savasan et al., Blood, 101:4449-4451 (2003);Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et al., Brit.J. Haemat 144:742-754 (2008)). Subjects with USS typically have 5-10% ofnormal ADAMTS13 activity (Kokame et al., PNAS 99(18):11902-11907, 2002).Although acquired TTP and USS have some similarities, USS has someimportant differences in clinical features. USS usually presents ininfancy or childhood and is characterized by severe hyperbilirubinemiawith negative Coombs test soon after birth, response to fresh plasmainfusion, and frequent relapses (Savasan et al., Blood, 101:4449-4451,2003). In some cases, patients with this inherited ADAMTS13 deficiencyhave a mild phenotype at birth and only develop symptoms associated withTTP in clinical situations with increased von Willebrand factor levels,such as infection or pregnancy. For example, Fujimura et al. reported 9Japanese women from 6 families with genetically confirmed USS who werediagnosed with the disorder during their first pregnancy.Thrombocytopenia occurred during the second to third trimesters in eachof their 15 pregnancies, often followed by TTP. All of these women werefound to be severely deficient in ADAMTS13 activity (Fujimura et al.,Brit. J. Haemat 144:742-754, 2008).

In accordance with the foregoing, in some embodiments, in the setting ofa subject with Upshaw-Schulman syndrome (USS) (i.e., the subject isknown to be deficient in ADAMTS13 activity and/or the subject is knownto have one or more ADAMTS13 gene mutation(s)), a method is provided fordecreasing the likelihood of developing clinical symptoms associatedwith congenital TTP (e.g., thrombocytopenia, anemia, fever, and/or renalfailure) comprising administering an amount of a MASP-2 inhibitory agent(e.g., a MASP-2 antibody) for a time period effective to ameliorate orprevent one or more clinical symptoms associated with TTP. In someembodiments, the method further comprises the step of determiningwhether a subject is at risk for developing symptoms associated withcongenital TTP prior to the onset of any symptoms associated with TTP,or upon the onset of at least one or more symptoms indicative of TTP(e.g., the presence of anemia, thrombocytopenia and/or renalinsufficiency). The determination of whether a subject is at risk fordeveloping symptoms associated with congenital TTP (i.e., the subjecthas USS), comprises determining whether the subject has a mutation inthe gene encoding ADAMTS13, and/or determining whether the subject isdeficient in ADAMTS13 activity, and/or determining whether the subjecthas a family history of USS. Methods of genetic screening for thepresence or absence of a genetic mutation associated with USS are wellestablished, for example see Kinoshita et al., International Journal ofHematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494(2001); Kokame et al., PNAS 99(18):11902-11907 (2002); Savasan et al.,Blood, 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310(2004) and Fujimura et al., Brit. J Haemat 144:742-754 (2008).

In one embodiment, a method is provided for reducing the likelihood thata subject diagnosed with USS will suffer clinical symptoms associatedwith TTP comprising periodically monitoring the subject to determine thepresence or absence of anemia, thrombocytopenia or rising creatinine,and treating with a MASP-2 inhibitory agent (e.g., a MASP-2 antibody)upon the determination of the presence of anemia, thrombocytopenia orrising creatinine, or upon the presence of an event known to beassociated with triggering TTP clinical symptoms, for example, drugexposure (e.g., chemotherapy), infection (e.g. bacterial infection),malignancy, injury, transplant, or pregnancy.

In another embodiment, a method is provided for treating a subject withUSS and suffering from clinical symptoms associated with TTP comprisingadministering an amount of a MASP-2 inhibitory agent (e.g., a MASP-2antibody) for a time period effective to ameliorate or prevent one ormore clinical symptoms associated with TTP.

TTP can also develop due to auto-antibodies against ADAMTS-13. Inaddition, TTP can develop during breast, gastrointestinal tract, orprostate cancer (George J N., Oncology (Williston Park). 25:908-14(2011)), pregnancy (second trimester or postpartum), George J N., CurrOpin Hematol 10:339-344 (2003)), or is associated with diseases, such asHIV or autoimmune diseases like systemic lupus erythematosis (HamasakiK, et al., Clin Rheumatol. 22:355-8 (2003)). TTP can also be caused bycertain drug therapies, including heparin, Quinine, immunemediatedingredient, cancer chemotherapeutic agents (bleomycin, cisplatin,cytosine arabinoside, daunomycin gemcitabine, mitomycin C, andtamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampinand anti-platelet drugs including ticlopidine and clopidogrel (Azarm, T.et al., J Res Med Sci., 16: 353-357 (2011)). Other factors or conditionsassociated with TTP are toxins such as bee venoms, sepsis, splenicsequestration, transplantation, vasculitis, vascular surgery, andinfections like Streptococcus pneumonia and cytomegalovirus (Moake J L.,N Engl J Med., 347:589-600 (2002)). TTP due to transient functionalADAMTS-13 deficiency can occur as a consequence of endothelial cellinjury associated with S. pneumonia infection (Pediatr Nephrol.,26:631-5 (2011)).

Plasma exchange is the standard treatment for TTP (Rock G A, et al.,NEngl J Med 325:393-397 (1991)). Plasma exchange replaces ADAMTS-13activity in patients with genetic defects and removes ADAMTS-13autoantibodies in those patients with acquired autoimmune TTP (Tsai,H-M, Hematol Oncol Clin North Am., 21(4): 609-v (2007)). Additionalagents such as immunosuppressive drugs are routinely added to therapy(George, J N, N Engl J Med, 354:1927-35 (2006)). However, plasmaexchange is not successful for about 20% of patients, relapse occurs inmore than a third of patients, and plasmapheresis is costly andtechnically demanding. Furthermore, many patients are unable to tolerateplasma exchange. Consequently there remains a critical need foradditional and better treatments for TTP.

Because TTP is a disorder of the blood coagulation cascade, treatmentwith antagonists of the complement system may aid in stabilizing andcorrecting the disease. While pathological activation of the alternativecomplement pathway is linked to aHUS, the role of complement activationin TTP is less clear. The functional deficiency of ADAMTS13 is importantfor the susceptibility of TTP, however it is not sufficient to causeacute episodes. Environmental factors and/or other genetic variationsmay contribute to the manifestation of TTP. For example, genes encodingproteins involved in the regulation of the coagulation cascade, vWF,platelet function, components of the endothelial vessel surface, or thecomplement system may be implicated in the development of acutethrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94:166-170 (2009)). In particular, complement activation has been shown toplay a critical role; serum from thrombotic microangiopathy associatedwith ADAMTS-13 deficiency has been shown to cause C3 and MAC depositionand subsequent neutrophil activation which could be abrogated bycomplement inactivation (Ruiz-Torres M P, et al., Thromb Haemost,93:443-52 (2005)). In addition, it has recently been shown that duringacute episodes of TTP there are increased levels of C4d, C3bBbP, and C3a(M. Reti et al., J Thromb Haemost. February 28 (2012) doi:10.1111/j.1538-7836.2012.04674.x. [Epub ahead of print]), consistentwith activation of the classical/lectin and alternative pathways. Thisincreased amount of complement activation in acute episodes may initiatethe terminal pathway activation and be responsible for furtherexacerbation of TTP.

The role of ADAMTS-13 and vWF in TTP clearly is responsible foractivation and aggregation of platelets and their subsequent role inshear stress and deposition in microangiopathies. Activated plateletsinteract with and trigger both the classical and alternative pathways ofcomplement. Platelet mediated complement activation increases theinflammatory mediators C3a and C5a (Peerschke E et al., Mol Immunol,47:2170-5 (2010)). Platelets may thus serve as targets of classicalcomplement activation in inherited or autoimmune TTP.

As described above, the lectin pathway of complement, by virtue ofMASP-2 mediated prothombin activation, is the dominant molecular pathwaylinking endothelial injury to the coagulation and microvascularthrombosis that occurs in HUS. Similarly, activation of the lectinpathway of complement may directly drive the coagulation system in TTP.Lectin pathway activation may be initiated in response to the initialendothelium injury caused by ADAMTS-13 deficiency in TTP. It istherefore expected that lectin pathway inhibitors, including but notlimited to antibodies that block MASP-2 function, will mitigate themicroangiopathies associated with microvascular coagulation, thrombosis,and hemolysis in patients suffering from TTP.

Patients suffering from TTP typically present in the emergency room withone or more of the following: purpura, renal failure, low platelets,anemia and/or thrombosis, including stroke. The current standard of carefor TTP involves intra-catheter delivery (e.g., intravenous or otherform of catheter) of replacement plasmapheresis for a period of twoweeks or longer, typically three times a week, but up to daily. If thesubject tests positive for the presence of an inhibitor of ADAMTS13(i.e., an endogenous antibody against ADAMTS13), then the plasmapheresismay be carried out in combination with immunosuppressive therapy (e.g.,corticosteroids, rituxan, or cyclosporine). Subjects with refractory TTP(approximately 20% of TTP patients) do not respond to at least two weeksof plasmapheresis therapy.

In accordance with the foregoing, in one embodiment, in the setting ofan initial diagnosis of TTP or in a subject exhibiting one or moresymptoms consistent with a diagnosis of TTP (e.g., central nervoussystem involvement, severe thrombocytopenia (a platelet count of lessthat or equal to 5000/μL if off aspirin, less than or equal to 20,000/μLif on aspirin), severe cardiac involvement, severe pulmonaryinvolvement, gastro-intestinal infarction or gangrene), a method isprovided for treating the subject with an effective amount of a MASP-2inhibitory agent (e.g., a anti-MASP-2 antibody) as a first line therapyin the absence of plasmapheresis, or in combination with plasmapheresis.As a first-line therapy, the MASP-2 inhibitory agent may be administeredto the subject systemically, such as by intra-arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration. In some embodiments, the MASP-2 inhibitory agent isadministered to a subject as a first-line therapy in the absence ofplasmapheresis to avoid the potential complications of plasmapheresis,such as hemorrhage, infection, and exposure to disorders and/orallergies inherent in the plasma donor, or in a subject otherwise averseto plasmapheresis, or in a setting where plasmapheresis is unavailable.In some embodiments, the MASP-2 inhibitory agent is administered to thesubject suffering from TTP in combination (including co-administration)with an immunosuppressive agent (e.g., corticosteroids, rituxan orcyclosporine) and/or in combination with concentrated ADAMTS-13.

In some embodiments, the method comprises administering a MASP-2inhibitory agent to a subject suffering from TTP via a catheter (e.g.,intravenously) for a first time period (e.g., an acute phase lasting atleast one day to a week or two weeks) followed by administering a MASP-2inhibitory agent to the subject subcutaneously for a second time period(e.g., a chronic phase of at least two weeks or longer). In someembodiments, the administration in the first and/or second time periodoccurs in the absence of plasmapheresis. In some embodiments, the methodis used to maintain the subject to prevent the subject from sufferingone or more symptoms associated with TTP.

In another embodiment, a method is provided for treating a subjectsuffering from refractory TTP (i.e., a subject that has not responded toat least two weeks of plasmaphoresis therapy), by administering anamount of a MASP-2 inhibitor effective to reduce one or more symptoms ofTTP. In one embodiment, the MASP-2 inhibitor (e.g., an anti-MASP-2antibody) is administered to a subject with refractory TTP on a chronicbasis, over a time period of at least two weeks or longer viasubcutaneous or other parenteral administration. Administration may berepeated as determined by a physician until the condition has beenresolved or is controlled.

In some embodiments, the method further comprises determining the levelof at least one complement factor (e.g., C3, C5) in the subject prior totreatment, and optionally during treatment, wherein the determination ofa reduced level of the at least one complement factor in comparison to astandard value or healthy control subject is indicative of the need forcontinued treatment with the MASP-2 inhibitory agent.

In some embodiments, the method comprises administering a MASP-2inhibitory agent, such as an anti-MASP-2 antibody, to a subjectsuffering from, or at risk for developing, TTP either subcutaneously orintravenously. Treatment is preferably daily, but can be as infrequentas biweekly. Treatment is continued until the subject's platelet countis greater than 150,000/ml for at least two consecutive days. Theanti-MASP-2 antibody may be administered alone, or in combination with aC5 inhibitor, such as eculizamab. In one embodiment, the MASP-2inhibitory antibody exhibits at least one or more of the followingcharacteristics: said antibody binds human MASP-2 with a K_(D) of 10 nMor less, said antibody binds an epitope in the CCP1 domain of MASP-2,said antibody inhibits C3b deposition in an in vitro assay in 1% humanserum at an IC₅₀ of 10 nM or less, said antibody inhibits C3b depositionin 90% human serum with an IC₅₀ of 30 nM or less, wherein the antibodyis an antibody fragment selected from the group consisting of Fv, Fab,Fab′, F(ab)₂ and F(ab′)₂, wherein the antibody is a single-chainmolecule, wherein said antibody is an IgG2 molecule, wherein saidantibody is an IgG1 molecule, wherein said antibody is an IgG4 molecule,wherein the IgG4 molecule comprises a S228P mutation, and/or wherein theantibody does not substantially inhibit the classical pathway. In oneembodiment, the antibody binds to MASP-2 and selectively inhibits thelectin pathway and does not substantially inhibit the alternativepathway. In one embodiment, the antibody binds to MASP-2 and selectivelyinhibits the lectin pathway and does not substantially inhibit theclassical pathway or the alternative pathway (i.e., inhibits the lectinpathway while leaving the classical and alternative complement pathwaysintact).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a subject suffering from TTP by at least 30%,such as at least 40%, such as at least 50%, such as at least 60%, suchas at least 70%, such as at least 80% such as at least 85%, such as atleast 90%, such as at least 95% up to 99%, as compared to untreatedserum. In some embodiments, the MASP-2 inhibitory antibody inhibitsthrombus formation in serum from a subject suffering from TTP at a levelof at least 20 percent or greater, (such as at least 30%, at least 40%,at least 50%) more than the inhibitory effect on C5b-9 deposition inserum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a TTP patient by at least 30%, such as at least40%, such as at least 50%, such as at least 60%, such as at least 70%,such as at least 80% such as at least 85%, such as at least 90%, such asat least 95% up to 99%, as compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to thesubject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibitingthrombus formation in a subject suffering from TTP comprisingadministering to the subject a composition comprising an amount of aMASP-2 inhibitory antibody, or antigen binding fragment thereof,comprising (I) (a) a heavy-chain variable region comprising: i) aheavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acidsequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) alight-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70, or (II) a variant thereofcomprising a heavy-chain variable region with at least 90% identity toSEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to SEQ ID NO:67) and a light-chain variable region with atleast 90% identity (e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a heavy-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:67. Insome embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a light-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, that specifically recognizes at least part of anepitope on human MASP-2 recognized by reference antibody OMS646comprising a heavy-chain variable region as set forth in SEQ ID NO:67and a light-chain variable region as set forth in SEQ ID NO:70.

Demos Disease

Degos disease, also known as malignant atrophic papulosis, is a veryrare TMA affecting the endothelium of small vessels of skin,gastrointestinal tract, and CNS. This vasculopathy causes occlusion ofvenules and artioles, resulting in skin lesions, bowel ischemia, and CNSdisorders including strokes, epilepsy and cognitive disorders. In theskin, connective tissue necrosis is due to thrombotic occlusion of thesmall arteries. However, the cause of Degos disease is unknown.Vasculitis, coagulopathy, or primary dysfunction of the endothelialcells have been implicated. Degos disease has a 50% survival of only twoto three years. There is no effective treatment for Degos diseasealthough antiplatelet drugs, anticoagulants, and immunosuppressants areutilized to alleviate symptoms.

While the mechanism of Degos disease is unknown, the complement pathwayhas been implicated. Margo et al., identified prominent C5b-9 depositsin skin, gastrointestinal tract and brain vessels of four terminalpatients with Degos disease (Margo et al., Am J Clin Pathol135(4):599-610, 2011). Experimental treatment with eculizumab wasinitially effective in the treatment of skin and intestinal lesions, butdid not prevent the progression of systemic disease (seeGarrett-Bakelman F. et al., “C5b-9 is a potential effector in thepathophysiology of Degos disease; a case report of treatment witheculizumab” (Abstract), Jerusalem: International Society of Hematology;2010, Poster #156; and Polito J. et al, “Early detection of systemicDegos disease (DD) or malignant atrophic papulosis (MAP) may increasesurvival” (Abstract), San Antonio, Tex.: American College ofGastroenterology; 2010, Poster #1205).

Many patients suffering from Degos disease have defects of bloodcoagulation. Thrombotic occlusion of small arteries in the skin ischaracteristic of the disease. Because the complement pathway isimplicated in this disease, as described herein for other TMAs, it isexpected that lectin pathway inhibitors, including but not limited toantibodies that block MASP-2 function, will be beneficial in treatingpatients suffering from Degos disease.

Accordingly, in another embodiment, the invention provides methods fortreating Degos disease by administering a composition comprising atherapeutically effective amount of a MASP-2 inhibitory agent, such as aMASP-2 antibody, in a pharmaceutical carrier to a subject suffering fromDegos disease or a condition resulting from Degos disease. The MASP-2inhibitory agent is administered systemically to the subject sufferingfrom Degos disease or a condition resulting from Degos disease, such asby intra-arterial, intravenous, intramuscular, inhalational,subcutaneous or other parenteral administration, or potentially by oraladministration for non-peptidergic agents. The anti-MASP-2 antibody maybe administered alone, or in combination with a C5 inhibitor, such aseculizamab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least oneor more of the following characteristics: said antibody binds humanMASP-2 with a K_(D) of 10 nM or less, said antibody binds an epitope inthe CCP1 domain of MASP-2, said antibody inhibits C3b deposition in anin vitro assay in 1% human serum at an IC₅₀ of 10 nM or less, saidantibody inhibits C3b deposition in 90% human serum with an IC₅₀ of 30nM or less, wherein the antibody is an antibody fragment selected fromthe group consisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)₂, wherein theantibody is a single-chain molecule, wherein said antibody is an IgG2molecule, wherein said antibody is an IgG molecule, wherein saidantibody is an IgG4 molecule, wherein the IgG4 molecule comprises aS228P mutation, and/or wherein the antibody does not substantiallyinhibit the classical pathway. In one embodiment, the antibody binds toMASP-2 and selectively inhibits the lectin pathway and does notsubstantially inhibit the alternative pathway. In one embodiment, theantibody binds to MASP-2 and selectively inhibits the lectin pathway anddoes not substantially inhibit the classical pathway or the alternativepathway (i.e., inhibits the lectin pathway while leaving the classicaland alternative complement pathways intact).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a subject suffering from Degos disease by atleast 30%, such as at least 40%, such as at least 50%, such as at least60%, such as at least 70%, such as at least 80% such as at least 85%,such as at least 90%, such as at least 95% up to 99%, as compared tountreated serum. In some embodiments, the MASP-2 inhibitory antibodyinhibits thrombus formation in serum from a subject suffering from Degosdisease at a level of at least 20 percent or greater, (such as at least30%, at least 40%, at least 50%) more than the inhibitory effect onC5b-9 deposition in serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a Degos disease patient by at least 30%, such asat least 40%, such as at least 50%, such as at least 60%, such as atleast 70%, such as at least 80% such as at least 85%, such as at least90%, such as at least 95% up to 99%, as compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to thesubject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibitingthrombus formation in a subject suffering from Degos disease comprisingadministering to the subject a composition comprising an amount of aMASP-2 inhibitory antibody, or antigen binding fragment thereof,comprising (I) (a) a heavy-chain variable region comprising: i) aheavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acidsequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) alight-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70, or (II) a variant thereofcomprising a heavy-chain variable region with at least 90% identity toSEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to SEQ ID NO:67) and a light-chain variable region with atleast 90% identity (e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a heavy-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:67. Insome embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a light-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, that specifically recognizes at least part of anepitope on human MASP-2 recognized by reference antibody OMS646comprising a heavy-chain variable region as set forth in SEQ ID NO:67and a light-chain variable region as set forth in SEQ ID NO:70.

Catastrophic Antiphospholipid Syndrome (CAPS)

Catastrophic antiphospholipid syndrome (CAPS) is an extreme variant ofthe antiphospholipid antibody (APLA) syndrome. CAPS is characterized byvenous and arterial thrombosis due to pathogenic antibodies. CAPS is aTMA with multiple organ thrombosis, ischemia, and organ failure. Likeother TMAs, occlusion of small vessels in various organs ischaracteristic. There is a high mortality rate in CAPS of about 50% andoften it is associated with infection or trauma. Patients haveantiphospholipid antibodies, generally IgG.

Clinically, CAPS involves at least three organs or tissues withhistopathological evidence of small vessel occlusion. Peripheralthrombosis may involve veins and arteries in the CNS, cardiovascular,renal, or pulmonary systems. Patients are treated with antibiotics,anticoagulants, corticosteroids, plasma exchange, and intravenousimmunoglobulin. Nevertheless, death may result from multiple organfailure.

The complement pathway has been implicated in CAPS. For example, studiesin animal models indicate that complement inhibition may be an effectivemeans to prevent thrombosis associated with CAPS (Shapira L. et al.,Arthritis Rheum 64(8):2719-23, 2012). Moreover, as further reported byShapira et al., administration of eculizumab to a subject suffering fromCAPS at doses that blocked complement pathway aborted acute progressivethrombotic events and reversed thrombocytopenia (see also Lim W., CurrOpin Hematol 18(5):361-5, 2011). Therefore, as described herein forother TMAs, it is expected that lectin pathway inhibitors, including butnot limited to antibodies that block MASP-2 function, will be beneficialin treating patients suffering from CAPS.

Accordingly, in another embodiment, the invention provides methods fortreating CAPS by administering a composition comprising atherapeutically effective amount of a MASP-2 inhibitory agent, such as aMASP-2 antibody, in a pharmaceutical carrier to a subject suffering fromCAPS or a condition resulting from CAPS. The MASP-2 inhibitory agent isadministered systemically to the subject suffering from CAPS or acondition resulting from CAPS, such as by intra-arterial, intravenous,intramuscular, inhalational, subcutaneous or other parenteraladministration, or potentially by oral administration fornon-peptidergic agents. The anti-MASP-2 antibody may be administeredalone, or in combination with a C5 inhibitor, such as eculizamab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least oneor more of the following characteristics: said antibody binds humanMASP-2 with a K_(D) of 10 nM or less, said antibody binds an epitope inthe CCP1 domain of MASP-2, said antibody inhibits C3b deposition in anin vitro assay in 1% human serum at an IC₅₀ of 10 nM or less, saidantibody inhibits C3b deposition in 90% human serum with an ICS0 of 30nM or less, wherein the antibody is an antibody fragment selected fromthe group consisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)₂, wherein theantibody is a single-chain molecule, wherein said antibody is an IgG2molecule, wherein said antibody is an IgG1 molecule, wherein saidantibody is an IgG4 molecule, wherein the IgG4 molecule comprises aS228P mutation, and/or wherein the antibody does not substantiallyinhibit the classical pathway. In one embodiment, the antibody binds toMASP-2 and selectively inhibits the lectin pathway and does notsubstantially inhibit the alternative pathway. In one embodiment, theantibody binds to MASP-2 and selectively inhibits the lectin pathway anddoes not substantially inhibit the classical pathway or the alternativepathway (i.e., inhibits the lectin pathway while leaving the classicaland alternative complement pathways intact).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a subject suffering from CAPS by at least 30%,such as at least 40%, such as at least 50%, such as at least 60%, suchas at least 70%, such as at least 80% such as at least 85%, such as atleast 90%, such as at least 95% up to 99%, as compared to untreatedserum. In some embodiments, the MASP-2 inhibitory antibody inhibitsthrombus formation in serum from a subject suffering from CAPS at alevel of at least 20 percent or greater, (such as at least 30%, at least40%, at least 50%) more than the inhibitory effect on C5b-9 depositionin serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a CAPS patient by at least 30%, such as at least40%, such as at least 50%, such as at least 60%, such as at least 70%,such as at least 80% such as at least 85%, such as at least 90%, such asat least 95% up to 99%, as compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to thesubject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibitingthrombus formation in a subject suffering from CAPS comprisingadministering to the subject a composition comprising an amount of aMASP-2 inhibitory antibody, or antigen binding fragment thereof,comprising (I) (a) a heavy-chain variable region comprising: i) aheavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acidsequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) alight-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70, or (II) a variant thereofcomprising a heavy-chain variable region with at least 90% identity toSEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to SEQ ID NO:67) and a light-chain variable region with atleast 90% identity (e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a heavy-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:67. Insome embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a light-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, that specifically recognizes at least part of anepitope on human MASP-2 recognized by reference antibody OMS646comprising a heavy-chain variable region as set forth in SEQ ID NO:67and a light-chain variable region as set forth in SEQ ID NO:70.

TMA Secondary to Cancer

Systemic malignancies of any type can lead to clinical and pathologicmanifestations of TMA (see e.g., Batts and Lazarus, Bone MarrowTransplantation 40:709-719, 2007). Cancer-associated TMA is often foundin the lungs and appears to be associated with tumor emboli (Francis K Ket al., Commun Oncol 2:339-43, 2005). Tumor emboli can reduce blood flowand thus lead to a hypo-perfused state in the affected arterioles andvenules. The resulting tissue stress and injury is expected to activatethe lectin pathway of complement in a localized fashion. The activatedlectin pathway in turn can activate the coagulation cascade via MASP-2dependent cleavage of prothrombin to thrombin, leading to apro-thrombotic state characteristic of TMA. MASP-2 inhibition in thissetting is expected to reduce the localized activation of thrombin andthereby alleviate the pro-thrombotic state.

Therefore, as described herein for other TMAs, it is expected thatlectin pathway inhibitors, including, but not limited to, antibodiesthat block MASP-2 function, will be beneficial in treating patientssuffering from TMA secondary to cancer.

Accordingly, in another embodiment, the invention provides methods fortreating or preventing TMA secondary to cancer by administering acomposition comprising a therapeutically effective amount of a MASP-2inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrierto a subject suffering from, or at risk for developing, a TMA secondaryto cancer. The MASP-2 inhibitory agent is administered systemically tothe subject suffering from, or at risk for developing, a TMA secondaryto cancer, such as by intra-arterial, intravenous, intramuscular,inhalational, subcutaneous or other parenteral administration, orpotentially by oral administration for non-peptidergic agents. Theanti-MASP-2 antibody may be administered alone, or in combination with aC5 inhibitor, such as eculizamab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least oneor more of the following characteristics: said antibody binds humanMASP-2 with a K_(D) of 10 nM or less, said antibody binds an epitope inthe CCP1 domain of MASP-2, said antibody inhibits C3b deposition in anin vitro assay in 1% human serum at an IC₅₀ of 10 nM or less, saidantibody inhibits C3b deposition in 90% human serum with an IC₅₀ of 30nM or less, wherein the antibody is an antibody fragment selected fromthe group consisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)₂, wherein theantibody is a single-chain molecule, wherein said antibody is an IgG2molecule, wherein said antibody is an IgG1 molecule, wherein saidantibody is an IgG4 molecule, wherein the IgG4 molecule comprises aS228P mutation, and/or wherein the antibody does not substantiallyinhibit the classical pathway. In one embodiment, the antibody binds toMASP-2 and selectively inhibits the lectin pathway and does notsubstantially inhibit the alternative pathway. In one embodiment, theantibody binds to MASP-2 and selectively inhibits the lectin pathway anddoes not substantially inhibit the classical pathway or the alternativepathway (i.e., inhibits the lectin pathway while leaving the classicaland alternative complement pathways intact).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a subject suffering from TMA secondary to cancerby at least 30%, such as at least 40%, such as at least 50%, such as atleast 60%, such as at least 70%, such as at least 80% such as at least85%, such as at least 90%, such as at least 95% up to 99%, as comparedto untreated serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a patient suffering TMA secondary to cancer byat least 30%, such as at least 40%, such as at least 50%, such as atleast 60%, such as at least 70%, such as at least 80% such as at least85%, such as at least 90%, such as at least 95% up to 99%, as comparedto untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to thesubject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibitingthrombus formation in a subject suffering from TMA secondary to cancercomprising administering to the subject a composition comprising anamount of a MASP-2 inhibitory antibody, or antigen binding fragmentthereof, comprising (I) (a) a heavy-chain variable region comprising: i)a heavy-chain CDR-H1 comprising the amino acid sequence from 31-35 ofSEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acidsequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) alight-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70, or (II) a variant thereofcomprising a heavy-chain variable region with at least 90% identity toSEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to SEQ ID NO:67) and a light-chain variable region with atleast 90% identity (e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a heavy-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:67. Insome embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a light-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, that specifically recognizes at least part of anepitope on human MASP-2 recognized by reference antibody OMS646comprising a heavy-chain variable region as set forth in SEQ ID NO:67and a light-chain variable region as set forth in SEQ ID NO:70.

TMA Secondary to Cancer Chemotherapy

Chemotherapy-associated TMA is a condition involving thrombocytopenia,microangiopathic hemolytic anemia, and renal dysfunction that developsin 2-10% of patients with a history of malignant neoplasms treated withchemotherapeutic agents such as gemcytabin, mitomycin, oxaliplatin andothers. Chemotherapy-associated TMA is associated with high mortalitypoor clinical outcomes (see, e.g., Blake-Haskins et al., Clin Cancer Res17(18):5858-5866, 2011).

The etiology of chemotherapy-associated TMA is thought to encompass anon-specific, toxic insult to the microvascular endothelium. A directinjury to endothelial cells has been shown in an animal model ofmitomycin-induced TMA (Dlott J. et al., Ther Apher Dial 8:102-11, 2004).Endothelial cell injury through a variety of mechanisms has been shownto activate the lectin pathway of complement. For example, Stahl et al.have shown that endothelial cells exposed to oxidative stress activatethe lectin pathway of complement both in vitro and in vivo (Collard etal., Am J Pathol. 156(5): 1549-56, 2000; La Bonte et al, J Immunol. 15;188(2):885-91, 2012). In vivo, this process leads to thombosis, andinhibition of the lectin pathway has been shown to prevent thrombosis(La Bonte et al. J Immunol. 15; 188(2):885-91, 2012). Futhermore, asdemonstrated in Examples 37-39 herein, in the mouse model of TMA wherelocalized photoexcitation of FITC-Dex was used to induce localizedinjury to the microvasculature with subsequent development of a TMAresponse, the present inventors have shown that inhibition of MASP-2 canprevent TMA. Thus, microvascular endothelium injury by chemotherapeuticagents may activate the lectin pathway of complement which then createsa localized pro-thrombotic state and promotes a TMA response. Sinceactivation of the lectin pathway and the creation of a pro-thomboticstate is MASP-2-dependent, it is expected that MASP-2 inhibitors,including, but not limited to, antibodies that block MASP-2 function,will alleviate the TMA response and reduce the risk of cancerchemotherapy-associated TMA.

Accordingly, in another embodiment, the invention provides methods fortreating or preventing TMA secondary to chemotherapy by administering acomposition comprising a therapeutically effective amount of a MASP-2inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrierto a subject suffering from, or at risk for developing, a TMA secondaryto chemotherapy. The MASP-2 inhibitory agent is administeredsystemically to a subject that has undergone, is undergoing, or willundergo chemotherapy, such as by intra-arterial, intravenous,intramuscular, inhalational, subcutaneous or other parenteraladministration, or potentially by oral administration fornon-peptidergic agents. The anti-MASP-2 antibody may be administeredalone, or in combination with a C5 inhibitor, such as eculizamab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least oneor more of the following characteristics: said antibody binds humanMASP-2 with a K_(D) of 10 nM or less, said antibody binds an epitope inthe CCP1 domain of MASP-2, said antibody inhibits C3b deposition in anin vitro assay in 1% human serum at an IC₅₀ of 10 nM or less, saidantibody inhibits C3b deposition in 90% human serum with an IC₅₀ of 30nM or less, wherein the antibody is an antibody fragment selected fromthe group consisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)₂, wherein theantibody is a single-chain molecule, wherein said antibody is an IgG2molecule, wherein said antibody is an IgG1 molecule, wherein saidantibody is an IgG4 molecule, wherein the IgG4 molecule comprises aS228P mutation, and/or wherein the antibody does not substantiallyinhibit the classical pathway. In one embodiment, the antibody binds toMASP-2 and selectively inhibits the lectin pathway and does notsubstantially inhibit the alternative pathway. In one embodiment, theantibody binds to MASP-2 and selectively inhibits the lectin pathway anddoes not substantially inhibit the classical pathway or the alternativepathway (i.e., inhibits the lectin pathway while leaving the classicaland alternative complement pathways intact).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a subject suffering from TMA secondary to cancerchemotherapy by at least 30%, such as at least 40%, such as at least50%, such as at least 60%, such as at least 70%, such as at least 80%such as at least 85%, such as at least 90%, such as at least 95% up to99%, as compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a patient suffering TMA secondary to cancerchemotherapy by at least 30%, such as at least 40%, such as at least50%, such as at least 60%, such as at least 70%, such as at least 80%such as at least 85%, such as at least 90%, such as at least 95% up to99%, as compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to thesubject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibitingthrombus formation in a subject suffering from TMA secondary to cancerchemotherapy comprising administering to the subject a compositioncomprising an amount of a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, comprising (I) (a) a heavy-chain variable regioncomprising: i) a heavy-chain CDR-H1 comprising the amino acid sequencefrom 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising theamino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chainCDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67and b) a light-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70, or (II) a variant thereofcomprising a heavy-chain variable region with at least 90% identity toSEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to SEQ ID NO:67) and a light-chain variable region with atleast 90% identity (e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a heavy-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:67. Insome embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a light-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, that specifically recognizes at least part of anepitope on human MASP-2 recognized by reference antibody OMS646comprising a heavy-chain variable region as set forth in SEQ ID NO:67and a light-chain variable region as set forth in SEQ ID NO:70.

TMA Secondary to Transplantation

Transplantation-associated TMA (TA-TMA) is a devastating syndrome thatcan occur in transplant patients, such as allogeneic hematopoietic stemcell transplant recipients (see e.g., Batts and Lazarus, Bone MarrowTransplantation 40:709-719, 2007). The pathogenesis of this condition ispoorly understood, but likely involves a confluence of responses thatculminate in endothelial cell injury (Laskin B. L. et al., Blood118(6):1452-62, 2011). As discussed above, endothelial cell injury is aprototypic stimulus for lectin pathway activation and the generation ofa pro-thrombotic environment.

Recent data further support the role of complement activation via thelectin pathway in the pathogenesis TA-TMA. Laskin et al., havedemonstrated that renal arteriolar C4d deposition was much more commonin subjects with histologic TA-TMA (75%) compared with controls (8%)(Laskin B. L., et al., Transplantation, 27; 96(2):217-23, 2013). Thus,C4d may be a pathologic marker of TA-TMA, implicating localizedcomplement fixation via the lectin or classical pathway.

Since activation of the lectin pathway and the creation of apro-thombotic state is MASP-2-dependent, it is expected that MASP-2inhibitors, including, but not limited to, antibodies that block MASP-2function, will alleviate the TMA response and reduce the risk oftransplantation-associated TMA (TA-TMA).

Accordingly, in another embodiment, the invention provides methods fortreating or preventing a TMA secondary to transplantation byadministering a composition comprising a therapeutically effectiveamount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in apharmaceutical carrier to a subject suffering from, or at risk fordeveloping a TMA secondary to transplantation. The MASP-2 inhibitoryagent is administered systemically to a subject that has undergone, isundergoing, or will undergo a transplant procedure, such as byintra-arterial, intravenous, intramuscular, inhalational, subcutaneousor other parenteral administration, or potentially by oraladministration for non-peptidergic agents. The anti-MASP-2 antibody maybe administered alone, or in combination with a C5 inhibitor, such aseculizamab. In some embodiments, the invention provides methods fortreating or preventing a TMA secondary to allogeneic stem celltransplant comprising administering a composition comprising an amountof a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to asubject prior to, during or after undergoing an allogeneic stem celltransplant.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least oneor more of the following characteristics: said antibody binds humanMASP-2 with a K_(D) of 10 nM or less, said antibody binds an epitope inthe CCP1 domain of MASP-2, said antibody inhibits C3b deposition in anin vitro assay in 1% human serum at an IC₅₀ of 10 nM or less, saidantibody inhibits C3b deposition in 90% human serum with an IC₅₀ of 30nM or less, wherein the antibody is an antibody fragment selected fromthe group consisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)₂, wherein theantibody is a single-chain molecule, wherein said antibody is an IgG2molecule, wherein said antibody is an IgG1 molecule, wherein saidantibody is an IgG4 molecule, wherein the IgG4 molecule comprises aS228P mutation, and/or wherein the antibody does not substantiallyinhibit the classical pathway. In one embodiment, the antibody binds toMASP-2 and selectively inhibits the lectin pathway and does notsubstantially inhibit the alternative pathway. In one embodiment, theantibody binds to MASP-2 and selectively inhibits the lectin pathway anddoes not substantially inhibit the classical pathway or the alternativepathway (i.e., inhibits the lectin pathway while leaving the classicaland alternative complement pathways intact).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a subject suffering from TMA secondary totransplant by at least 30%, such as at least 40%, such as at least 50%,such as at least 60%, such as at least 70%, such as at least 80% such asat least 85%, such as at least 90%, such as at least 95% up to 99%, ascompared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a patient suffering TMA secondary to transplantby at least 30%, such as at least 40%, such as at least 50%, such as atleast 60%, such as at least 70%, such as at least 80% such as at least85%, such as at least 90%, such as at least 95% up to 99%, as comparedto untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to thesubject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibitingthrombus formation in a subject suffering from TMA secondary totransplant comprising administering to the subject a compositioncomprising an amount of a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, comprising (I) (a) a heavy-chain variable regioncomprising: i) a heavy-chain CDR-H1 comprising the amino acid sequencefrom 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising theamino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chainCDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67and b) a light-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70, or (II) a variant thereofcomprising a heavy-chain variable region with at least 90% identity toSEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to SEQ ID NO:67) and a light-chain variable region with atleast 90% identity (e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a heavy-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:67. Insome embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a light-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, that specifically recognizes at least part of anepitope on human MASP-2 recognized by reference antibody OMS646comprising a heavy-chain variable region as set forth in SEQ ID NO:67and a light-chain variable region as set forth in SEQ ID NO:70.

IV. THE ROLE OF MASP-2 IN OTHER DISEASES AND CONDITIONS AND THERAPEUTICMETHODS USING MASP-2 INHIBITORY AGENTS

Renal Conditions

Activation of the complement system has been implicated in thepathogenesis of a wide variety of renal diseases; including,mesangioproliferative glomerulonephritis (IgA-nephropathy, Berger'sdisease) (Endo, M., et al., Clin. Nephrology 55:185-191, 2001),membranous glomerulonephritis (Kerjashki, D., Arch B Cell Pathol.58:253-71, 1990; Brenchley, P. E., et al., Kidney Int., 41:933-7, 1992;Salant, D. J., et al., Kidney Int. 35:976-84, 1989),membranoproliferative glomerulonephritis (mesangiocapillaryglomerulonephritis) (Bartlow, B. G., et al., Kidney Int. 15:294-300,1979; Meri, S., et al., J. Exp. Med. 175:939-50, 1992), acutepostinfectious glomerulonephritis (poststreptococcalglomerulonephritis), cryoglobulinemic glomerulonephritis (Ohsawa, I., etal., Clin Immunol. 101:59-66, 2001), lupus nephritis (Gatenby, P. A.,Autoimmunity 11:61-6, 1991), and Henoch-Schonlein purpura nephritis(Endo, M., et al., Am. J. Kidney Dis. 35:401-407, 2000). The involvementof complement in renal disease has been appreciated for several decadesbut there is still a major discussion on its exact role in the onset,the development and the resolution phase of renal disease. Under normalconditions the contribution of complement is beneficial to the host, butinappropriate activation and deposition of complement may contribute totissue damage.

There is substantial evidence that glomerulonephritis, inflammation ofthe glomeruli, is often initiated by deposition of immune complexes ontoglomerular or tubular structures which then triggers complementactivation, inflammation and tissue damage. Kahn and Sinniahdemonstrated increased deposition of C5b-9 in tubular basement membranesin biopsies taken from patients with various forms of glomerulonephritis(Kahn, T. N., et al., Histopath. 26:351-6, 1995). In a study of patientswith IgA nephrology (Alexopoulos, A., et al., Nephrol. Dial. Transplant10:1166-1172, 1995), C5b-9 deposition in the tubular epithelial/basementmembrane structures correlated with plasma creatinine levels. Anotherstudy of membranous nephropathy demonstrated a relationship betweenclinical outcome and urinary sC5b-9 levels (Kon, S. P., et al., KidneyInt. 48:1953-58, 1995). Elevated sC5b-9 levels were correlatedpositively with poor prognosis. Lehto et al., measured elevated levelsof CD59, a complement regulatory factor that inhibits the membraneattack complex in plasma membranes, as well as C5b-9 in urine frompatients with membranous glomerulonephritis (Lehto, T., et al., KidneyInt. 47:1403-11, 1995). Histopathological analysis of biopsy samplestaken from these same patients demonstrated deposition of C3 and C9proteins in the glomeruli, whereas expression of CD59 in these tissueswas diminished compared to that of normal kidney tissue. These variousstudies suggest that ongoing complement-mediated glomerulonephritisresults in urinary excretion of complement proteins that correlate withthe degree of tissue damage and disease prognosis.

Inhibition of complement activation in various animal models ofglomerulonephritis has also demonstrated the importance of complementactivation in the etiology of the disease. In a model ofmembranoproliferative glomerulonephritis (MPGN), infusion of anti-Thy1antiserum in C6-deficient rats (that cannot form C5b-9) resulted in 90%less glomerular cellular proliferation, 80% reduction in platelet andmacrophage infiltration, diminished collagen type IV synthesis (a markerfor mesangial matrix expansion), and 50% less proteinuria than inC6+normal rats (Brandt, J., et al., Kidney Int. 49:335-343, 1996). Theseresults implicate C5b-9 as a major mediator of tissue damage bycomplement in this rat anti-thymocyte serum model. In another model ofglomerulonephritis, infusion of graded dosages of rabbit anti-ratglomerular basement membrane produced a dose-dependent influx ofpolymorphonuclear leukocytes (PMN) that was attenuated by priortreatment with cobra venom factor (to consume complement) (Scandrett, A.L., et al., Am. J. Physiol. 268:F256-F265, 1995). Cobra venomfactor-treated rats also showed diminished histopathology, decreasedlong-term proteinuria, and lower creatinine levels than control rats.Employing three models of GN in rats (anti-thymocyte serum, Con Aanti-Con A, and passive Heymann nephritis), Couser et al., demonstratedthe potential therapeutic efficacy of approaches to inhibit complementby using the recombinant sCRI protein (Couser, W. G., et al., J. Am.Soc. Nephrol. 5:1888-94, 1995). Rats treated with sCRI showedsignificantly diminished PMN, platelet and macrophage influx, decreasedmesangiolysis, and proteinuria versus control rats. Further evidence forthe importance of complement activation in glomerulonephritis has beenprovided by the use of an anti-C5 MoAb in the NZB/W F1 mouse model. Theanti-C5 MoAb inhibits cleavage of C5, thus blocking generation of C5aand C5b-9. Continuous therapy with anti-C5 MoAb for 6 months resulted insignificant amelioration of the course of glomerulonephritis. Ahumanized anti-C5 MoAb monoclonal antibody (5G1.1) that prevents thecleavage of human complement component C5 into its pro-inflammatorycomponents is under development by Alexion Pharmaceuticals, Inc., NewHaven, Conn., as a potential treatment for glomerulonephritis.

Direct evidence for a pathological role of complement in renal injury isprovided by studies of patients with genetic deficiencies in specificcomplement components. A number of reports have documented anassociation of renal disease with deficiencies of complement regulatoryfactor H (Ault, B. H., Nephrol. 14:1045-1053, 2000; Levy, M., et al.,Kidney Int. 30:949-56, 1986; Pickering, M. C., et al., Nat. Genet.31:424-8, 2002). Factor H deficiency results in low plasma levels offactor B and C3 and in consumption of C5b-9. Both atypicalmembranoproliferative glomerulonephritis (MPGN) and idiopathic hemolyticuremic syndrome (HUS) are associated with factor H deficiency. Factor Hdeficient pigs (Jansen, J. H., et al., Kidney Int. 53:331-49, 1998) andfactor H knockout mice (Pickering, M. C., 2002) display MPGN-likesymptoms, confirming the importance of factor H in complementregulation. Deficiencies of other complement components are associatedwith renal disease, secondary to the development of systemic lupuserythematosus (SLE) (Walport, M. J., Davies, et al., Ann. N.Y. Acad.Sci. 815:267-81, 1997). Deficiency for Clq, C4 and C2 predisposestrongly to the development of SLE via mechanisms relating to defectiveclearance of immune complexes and apoptotic material. In many of theseSLE patients lupus nephritis occurs, characterized by the deposition ofimmune complexes throughout the glomerulus.

Further evidence linking complement activation and renal disease hasbeen provided by the identification in patients of autoantibodiesdirected against complement components, some of which have been directlyrelated to renal disease (Trouw, L. A., et al., Mol. Immunol.38:199-206, 2001). A number of these autoantibodies show such a highdegree of correlation with renal disease that the term nephritic factor(NeF) was introduced to indicate this activity. In clinical studies,about 50% of the patients positive for nephritic factors developed MPGN(Spitzer, R. E., et al., Clin. Immunol. Immunopathol. 64:177-83, 1992).C3NeF is an autoantibody directed against the alternative pathway C3convertase (C3bBb) and it stabilizes this convertase, thereby promotingalternative pathway activation (Daha, M. R., et al., J. Immunol.116:1-7, 1976). Likewise, autoantibody with a specificity for theclassical pathway C3 convertase (C4b2a), called C4NeF, stabilizes thisconvertase and thereby promotes classical pathway activation (Daha, M.R. et al., J. Immunol. 125:2051-2054, 1980; Halbwachs, L., et al., J.Clin. Invest. 65:1249-56, 1980). Anti-C1q autoantibodies have beendescribed to be related to nephritis in SLE patients (Hovath, L., etal., Clin. Exp. Rheumatol. 19:667-72, 2001; Siegert, C., et al., J.Rheumatol. 18:230-34, 1991; Siegert, C., et al., Clin. Exp. Rheumatol.10:19-23, 1992), and a rise in the titer of these anti-Clqautoantibodies was reported to predict a flare of nephritis (Coremans,I. E., et al., Am. J. Kidney Dis. 26:595-601, 1995). Immune depositseluted from postmortem kidneys of SLE patients revealed the accumulationof these anti-Clq autoantibodies (Mannick, M., et al., ArthritisRheumatol. 40:1504-11, 1997). All these facts point to a pathologicalrole for these autoantibodies. However, not all patients with anti-Clqautoantibodies develop renal disease and also some healthy individualshave low titer anti-Clq autoantibodies (Siegert, C. E., et al., Clin.Immunol. Immunopathol. 67:204-9, 1993).

In addition to the alternative and classical pathways of complementactivation, the lectin pathway may also have an important pathologicalrole in renal disease. Elevated levels of MBL, MBL-associated serineprotease and complement activation products have been detected byimmunohistochemical techniques on renal biopsy material obtained frompatients diagnosed with several different renal diseases, includingHenoch-Schonlein purpura nephritis (Endo, M., et al., Am. J. Kidney Dis.35:401-407, 2000), cryoglobulinemic glomerulonephritis (Ohsawa, I., etal., Clin. Immunol. 101:59-66, 2001) and IgA neuropathy (Endo, M., etal., Clin. Nephrology 55:185-191, 2001). Therefore, despite the factthat an association between complement and renal diseases has been knownfor several decades, data on how complement exactly influences theserenal diseases is far from complete.

Blood Disorders

Sepsis is caused by an overwhelming reaction of the patient to invadingmicroorganisms. A major function of the complement system is toorchestrate the inflammatory response to invading bacteria and otherpathogens. Consistent with this physiological role, complementactivation has been shown in numerous studies to have a major role inthe pathogenesis of sepsis (Bone, R. C., Annals. Internal. Med.115:457-469, 1991). The definition of the clinical manifestations ofsepsis is ever evolving. Sepsis is usually defined as the systemic hostresponse to an infection. However, on many occasions, no clinicalevidence for infection (e.g., positive bacterial blood cultures) isfound in patients with septic symptoms. This discrepancy was first takeninto account at a Consensus Conference in 1992 when the term “systemicinflammatory response syndrome” (SIRS) was established, and for which nodefinable presence of bacterial infection was required (Bone, R. C., etal., Crit. Care Med. 20:724-726, 1992). There is now general agreementthat sepsis and SIRS are accompanied by the inability to regulate theinflammatory response. For the purposes of this brief review, we willconsider the clinical definition of sepsis to also include severesepsis, septic shock, and SIRS.

The predominant source of infection in septic patients before the late1980s was Gram-negative bacteria. Lipopolysaccharide (LPS), the maincomponent of the Gram-negative bacterial cell wall, was known tostimulate release of inflammatory mediators from various cell types andinduce acute infectious symptoms when injected into animals (Haeney, M.R., et al., Antimicrobial Chemotherapy 41(Suppl. A):41-6, 1998).Interestingly, the spectrum of responsible microorganisms appears tohave shifted from predominantly Gram-negative bacteria in the late 1970sand 1980s to predominantly Gram-positive bacteria at present, forreasons that are currently unclear (Martin, G. S., et al., N. Eng. J.Med. 348:1546-54, 2003).

Many studies have shown the importance of complement activation inmediating inflammation and contributing to the features of shock,particularly septic and hemorrhagic shock. Both Gram-negative andGram-positive organisms commonly precipitate septic shock. LPS is apotent activator of complement, predominantly via the alternativepathway, although classical pathway activation mediated by antibodiesalso occurs (Fearon, D. T., et al., N. Engl. J. Med. 292:937-400, 1975).The major components of the Gram-positive cell wall are peptidoglycanand lipoteichoic acid, and both components are potent activators of thealternative complement pathway, although in the presence of specificantibodies they can also activate the classical complement pathway(Joiner, K. A., et al., Ann. Rev. Immunol. 2:461-2, 1984).

The complement system was initially implicated in the pathogenesis ofsepsis when it was noted by researchers that anaphylatoxins C3a and C5amediate a variety of inflammatory reactions that might also occur duringsepsis. These anaphylatoxins evoke vasodilation and an increase inmicrovascular permeability, events that play a central role in septicshock (Schumacher, W. A., et al., Agents Actions 34:345-349, 1991). Inaddition, the anaphylatoxins induce bronchospasm, histamine release frommast cells, and aggregation of platelets. Moreover, they exert numerouseffects on granulocytes, such as chemotaxis, aggregation, adhesion,release of lysosomal enzymes, generation of toxic super oxide anion andformation of leukotrienes (Shin, H. S., et al., Science 162:361-363,1968; Vogt, W., Complement 3:177-86, 1986). These biologic effects arethought to play a role in development of complications of sepsis such asshock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, D.E., et al., Lancet 1:947-949, 1980; Slotman, G. T., et al., Surgery99:744-50, 1986). Furthermore, elevated levels of the anaphylatoxin C3ais associated with a fatal outcome in sepsis (Hack, C. E., et al., Am.J. Med. 86:20-26, 1989). In some animal models of shock, certaincomplement-deficient strains (e.g., C5-deficient ones) are moreresistant to the effects of LPS infusions (Hseuh, W., et al., Immunol.70:309-14, 1990).

Blockade of C5a generation with antibodies during the onset of sepsis inrodents has been shown to greatly improve survival (Czermak, B. J., etal., Nat. Med. 5:788-792, 1999). Similar findings were made when the C5areceptor (CSaR) was blocked, either with antibodies or with a smallmolecular inhibitor (Huber-Lang, M. S., et al., FASEB J. 16:1567-74,2002; Riedemann, N.C., et al., J. Clin. Invest. 110:101-8, 2002).Earlier experimental studies in monkeys have suggested that antibodyblockade of C5a attenuated E. coli-induced septic shock and adultrespiratory distress syndrome (Hangen, D. H., et al., J. Surg. Res.46:195-9, 1989; Stevens, J. H., et al., J. Clin. Invest. 77:1812-16,1986). In humans with sepsis, C5a was elevated and associated withsignificantly reduced survival rates together with multiorgan failure,when compared with that in less severely septic patients and survivors(Nakae, H., et al., Res. Commun. Chem. Pathol. Pharmacol. 84:189-95,1994; Nakae, et al., Surg. Today 26:225-29, 1996; Bengtson, A., et al.,Arch. Surg. 123:645-649, 1988). The mechanisms by which C5a exerts itsharmful effects during sepsis are yet to be investigated in greaterdetail, but recent data suggest the generation of C5a during sepsissignificantly compromises innate immune functions of blood neutrophils(Huber-Lang, M. S., et al., J. Immunol. 169:3223-31, 2002), theirability to express a respiratory burst, and their ability to generatecytokines (Riedemann, N.C., et al., Immunity 19:193-202, 2003). Inaddition, C5a generation during sepsis appears to have procoagulanteffects (Laudes, I. J., et al., Am. J. Pathol. 160:1867-75, 2002). Thecomplement-modulating protein CI INH has also shown efficacy in animalmodels of sepsis and ARDS (Dickneite, G., Behring Ins. Mitt. 93:299-305,1993).

The lectin pathway may also have a role in pathogenesis of sepsis. MBLhas been shown to bind to a range of clinically important microorganismsincluding both Gram-negative and Gram-positive bacteria, and to activatethe lectin pathway (Neth, O., et al., Infect. Immun. 68:688, 2000).Lipoteichoic acid (LTA) is increasingly regarded as the Gram-positivecounterpart of LPS. It is a potent immunostimulant that induces cytokinerelease from mononuclear phagocytes and whole blood (Morath, S., et al.,J. Exp. Med. 195:1635, 2002; Morath, S., et al., Infect. Immun. 70:938,2002). Recently it was demonstrated that L-ficolin specifically binds toLTA isolated from numerous Gram-positive bacteria species, includingStaphylococcus aureus, and activates the lectin pathway (Lynch, N. J.,et al., J. Immunol. 172:1198-02, 2004). MBL also has been shown to bindto LTA from Enterococcus spp in which the polyglycerophosphate chain issubstituted with glycosyl groups), but not to LTA from nine otherspecies including S. aureus (Polotsky, V. Y., et al., Infect. Immun.64:380, 1996).

An aspect of the invention thus provides a method for treating sepsis ora condition resulting from sepsis, by administering a compositioncomprising a therapeutically effective amount of a MASP-2 inhibitoryagent in a pharmaceutical carrier to a subject suffering from sepsis ora condition resulting from sepsis including without limitation severesepsis, septic shock, acute respiratory distress syndrome resulting fromsepsis, and systemic inflammatory response syndrome. Related methods areprovided for the treatment of other blood disorders, includinghemorrhagic shock, hemolytic anemia, autoimmune thromboticthrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS),atypical hemolytic uremic syndrome (aHUS), or other marrow/blooddestructive conditions, by administering a composition comprising atherapeutically effective amount of a MASP-2 inhibitory agent in apharmaceutical carrier to a subject suffering from such a condition. TheMASP-2 inhibitory agent is administered to the subject systemically,such as by intra-arterial, intravenous, intramuscular, inhalational(particularly in the case of ARDS), subcutaneous or other parenteraladministration, or potentially by oral administration fornon-peptidergic agents. The MASP-2 inhibitory agent composition may becombined with one or more additional therapeutic agents to combat thesequelae of sepsis and/or shock. For advanced sepsis or shock or adistress condition resulting therefrom, the MASP-2 inhibitorycomposition may suitably be administered in a fast-acting dosage form,such as by intravenous or intra-arterial delivery of a bolus of asolution containing the MASP-2 inhibitory agent composition. Repeatedadministration may be carried out as determined by a physician until thecondition has been resolved.

Coagulopathies

Evidence has been developed for the role of the complement system indisseminated intravascular coagulation (“DIC”), such as DIC secondary tosignificant bodily trauma.

Previous studies have shown that C4−/− mice are not protected from renalreperfusion injury. (Zhou, W., et al, “Predominant role for C5b-9 inrenal ischemia/reperfusion injury,” J Clin Invest 105:1363-1371 (2000))In order to investigate whether C4−/− mice may still be able to activatecomplement via either the classical or the lectin pathway, C3 turn-overin C4−/− plasma was measured in assays specific for either theclassical, or the lectin pathway activation route. While no C3 cleavagecould be observed when triggering activation via the classical, a highlyefficient lectin pathway-dependent activation of C3 in C4 deficientserum was observed (FIG. 30). It can be seen that C3b deposition onmannan and zymosan is severely compromised in MASP-2−/− mice, even underexperimental conditions, that according to many previously publishedpapers on alternative pathway activation, should be permissive for allthree pathways. When using the same sera in wells coated withimmunoglobulin complexes instead of mannan or zymosan, C3b depositionand Factor B cleavage are seen in MASP-2+/+mouse sera and MASP-2−/−sera, but not in C1q depleted sera. This indicates that alternatepathway activation is facilitated in MASP-2−/− sera when the initial C3bis provided via classical activity. FIG. 30C depicts the surprisingfinding that C3 can efficiently be activated in a lectinpathway-dependent fashion in C4 deficient plasma.

This “C4 bypass” is abolished by the inhibition of lectinpathway-activation through preincubation of plasma with soluble mannanor mannose.

Aberrant, non-immune, activation of the complement system is potentiallyhazardous to man and may also play an important role in hematologicalpathway activation, particularly in severe trauma situations whereinboth inflammatory and hematological pathways are activated. In normalhealth, C3 conversion is <5% of the total plasma C3 protein. In rampantinfection, including septicaemia and immune complex disease, C3conversion re-establishes itself at about 30% with complement levelsfrequently lower than normal, due to increased utilization and changesin pool distribution. Immediate C3 pathway activation of greater than30% generally produces obvious clinical evidence of vasodilatation andof fluid loss to the tissues. Above 30% C3 conversion, the initiatingmechanisms are predominantly non-immune and the resulting clinicalmanifestations are harmful to the patient. Complement C5 levels inhealth and in controlled disease appear much more stable than C3.Significant decreases and or conversion of C5 levels are associated withthe patient's response to abnormal polytrauma (e.g., road trafficaccidents) and the likely development of shock lung syndromes. Thus, anyevidence of either complement C3 activation beyond 30% of the vascularpool or of any C5 involvement, or both, may be considered likely to be aharbinger of a harmful pathological change in the patient.

Both C3 and C5 liberate anaphylatoxins (C3a and C5a) that act on mastcells and basophils releasing vasodilatory chemicals. They set upchemotactic gradients to guide polymorphonuclear cells (PMN) to thecenter of immunological disturbances (a beneficial response), but herethey differ because C5a has a specific clumping (aggregating) effect onthese phagocytic cells, preventing their random movement away from thereaction site. In normal control of infection, C3 activates C5. However,in polytrauma, C5 appears to be widely activated, generating C5aanaphylatoxins systemically. This uncontrolled activity causespolymorphs to clump within the vascular system, and these clumps arethen swept into the capillaries of the lungs, which they occlude andgenerate local damaging effects as a result of superoxide liberation.While not wishing to be limited by theory, the mechanism is probablyimportant in the pathogenesis of acute respiratory distress syndrome(ARDS), although this view has recently been challenged. The C3aanaphylatoxins in vitro can be shown to be potent platelet aggregators,but their involvement in vivo is less defined and the release ofplatelet substances and plasmin in wound repair may only secondarilyinvolve complement C3. It is possible that prolonged elevation of C3activation is necessary to generate DIC.

In addition to cellular and vascular effects of activated complementcomponent outlined above that could explain the link between trauma andDIC, emerging scientific discoveries have identified direct molecularlinks and functional cross-talk between complement and coagulationsystems. Supporting data has been obtained from studies in C3 deficientmice. Because C3 is the shared component for each of the complementpathways, C3 deficient mice are predicted to lack all complementfunction. Surprisingly, however, C3 deficient mice are perfectly capableof activating terminal complement components. (Huber-Lang, M., et al.,“Generation of C5a in the absence of C3: a new complement activationpathway,” Nat. Med 12:682-687 (2006)) In depth studies revealed thatC3-independent activation of terminal complement components is mediatedby thrombin, the rate limiting enzyme of the coagulation cascade. (Huberet al., 2006) The molecular components mediating thrombin activationfollowing initial complement activation remained elusive.

The present inventors have elucidated what is believed to be themolecular basis for cross-talk between complement and clotting cascadesand identified MASP-2 as a central control point linking the twosystems. Biochemical studies into the substrate specificity of MASP-2have identified prothrombin as a possible substrate, in addition to thewell known C2 and C4 complement proteins. MASP-2 specifically cleavesprothrombin at functionally relevant sites, generating thrombin, therate limiting enzyme of the coagulation cascade. (Krarup, A., et al.,“Simultaneous Activation of Complement and Coagulation by MBL-AssociatedSerine Protease 2,” PLoS. ONE. 2:e623 (2007)) MASP-2-generated thrombinis capable of promoting fibrin deposition in a defined reconstituted invitro system, demonstrating the functional relevance of MASP-2 cleavage.(Krarup et al., 2007) As discussed in the examples herein below, theinventors have further corroborated the physiological significance ofthis discovery by documenting thrombin activation in normal rodent serumfollowing lectin pathway activation, and demonstrated that this processis blocked by neutralizing MASP-2 monoclonal antibodies.

MASP-2 may represent a central branch point in the lectin pathway,capable of promoting activation of both complement and coagulationsystems. Because lectin pathway activation is a physiologic response tomany types of traumatic injury, the present inventors believe thatconcurrent systemic inflammation (mediated by complement components) anddisseminated coagulation (mediated via the clotting pathway) can beexplained by the capacity of MASP-2 to activate both pathways. Thesefindings clearly suggest a role for MASP-2 in DIC generation andtherapeutic benefit of MASP-2 inhibition in treating or preventing DIC.MASP-2 may provide the molecular link between complement and coagulationsystem, and activation of the lectin pathway as it occurs in settings oftrauma can directly initiate activation of the clotting system via theMASP-2-thrombin axis, providing a mechanistic link between trauma andDIC. In accordance with an aspect of the present invention, inhibitionof MASP-2 would inhibit lectin pathway activation and reduce thegeneration of both anaphylatoxins C3a and C5a. It is believed thatprolonged elevation of C3 activation is necessary to generate DIC.

Microcirculatory coagulation (blot clots in capillaries and small bloodvessels) occurs in settings such a septic shock. A role of the lectinpathway in septic shock is established, as evidenced by the protectedphenotype of MASP-2 (−/−) mouse models of sepsis, described in Example17 and FIGS. 18 and 19. Furthermore, as demonstrated in Example 15 andFIGS. 16A and 16B, MASP-2 (−/−) mice are protected in the localizedSchwartzman reaction model of disseminated intravascular coagulation(DIC), a model of localized coagulation in microvessels.

V. MASP-2 INHIBITORY AGENTS

In one aspect, the present invention provides methods of inhibitingMASP-2-dependent complement activation in a subject suffering from, orat risk for developing a thrombotic microangiopathy. MASP-2 inhibitoryagents are administered in an amount effective to inhibitMASP-2-dependent complement activation in a living subject. In thepractice of this aspect of the invention, representative MASP-2inhibitory agents include: molecules that inhibit the biologicalactivity of MASP-2 (such as small molecule inhibitors, anti-MASP-2antibodies or blocking peptides which interact with MASP-2 or interferewith a protein-protein interaction), and molecules that decrease theexpression of MASP-2 (such as MASP-2 antisense nucleic acid molecules,MASP-2 specific RNAi molecules and MASP-2 ribozymes), thereby preventingMASP-2 from activating the lectin complement pathway. The MASP-2inhibitory agents can be used alone as a primary therapy or incombination with other therapeutics as an adjuvant therapy to enhancethe therapeutic benefits of other medical treatments.

The inhibition of MASP-2-dependent complement activation ischaracterized by at least one of the following changes in a component ofthe complement system that occurs as a result of administration of aMASP-2 inhibitory agent in accordance with the methods of the invention:the inhibition of the generation or production of MASP-2-dependentcomplement activation system products C4b, C3a, C5a and/or C5b-9 (MAC)(measured, for example, as described in Example 2), the reduction ofcomplement activation assessed in a hemolytic assay using unsensitizedrabbit or guinea pig red blood cells (measured, for example as describedin Example 33), the reduction of C4 cleavage and C4b deposition(measured, for example as described in Example 2), or the reduction ofC3 cleavage and C3b deposition (measured, for example, as described inExample 2).

According to the present invention, MASP-2 inhibitory agents areutilized that are effective in inhibiting the MASP-2-dependentcomplement activation system. MASP-2 inhibitory agents useful in thepractice of this aspect of the invention include, for example,anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibitorypeptides, small molecules, MASP-2 soluble receptors and expressioninhibitors. MASP-2 inhibitory agents may inhibit the MASP-2-dependentcomplement activation system by blocking the biological function ofMASP-2. For example, an inhibitory agent may effectively block MASP-2protein-to-protein interactions, interfere with MASP-2 dimerization orassembly, block Ca²⁺ binding, interfere with the MASP-2 serine proteaseactive site, or may reduce MASP-2 protein expression.

In some embodiments, the MASP-2 inhibitory agents selectively inhibitMASP-2 complement activation, leaving the Clq-dependent complementactivation system functionally intact.

In one embodiment, a MASP-2 inhibitory agent useful in the methods ofthe invention is a specific MASP-2 inhibitory agent that specificallybinds to a polypeptide comprising SEQ ID NO:6 with an affinity of atleast ten times greater than to other antigens in the complement system.In another embodiment, a MASP-2 inhibitory agent specifically binds to apolypeptide comprising SEQ ID NO:6 with a binding affinity of at least100 times greater than to other antigens in the complement system. Thebinding affinity of the MASP-2 inhibitory agent can be determined usinga suitable binding assay.

The MASP-2 polypeptide exhibits a molecular structure similar to MASP-1,MASP-3, and C1r and C1s, the proteases of the C1 complement system. ThecDNA molecule set forth in SEQ ID NO:4 encodes a representative exampleof MASP-2 (consisting of the amino acid sequence set forth in SEQ IDNO:5) and provides the human MASP-2 polypeptide with a leader sequence(aa 1-15) that is cleaved after secretion, resulting in the mature formof human MASP-2 (SEQ ID NO:6). As shown in FIG. 2, the human MASP 2 geneencompasses twelve exons. The human MASP-2 cDNA is encoded by exons B,C, D, F, G, H, I, J, K AND L. An alternative splice results in a 20 kDaprotein termed MBL-associated protein 19 (“MAp19”, also referred to as“sMAP”) (SEQ ID NO:2), encoded by (SEQ ID NO:1) arising from exons B, C,D and E as shown in FIG. 2. The cDNA molecule set forth in SEQ ID NO:50encodes the murine MASP-2 (consisting of the amino acid sequence setforth in SEQ ID NO:51) and provides the murine MASP-2 polypeptide with aleader sequence that is cleaved after secretion, resulting in the matureform of murine MASP-2 (SEQ ID NO:52). The cDNA molecule set forth in SEQID NO:53 encodes the rat MASP-2 (consisting of the amino acid sequenceset forth in SEQ ID NO:54) and provides the rat MASP-2 polypeptide witha leader sequence that is cleaved after secretion, resulting in themature form of rat MASP-2 (SEQ ID NO:55).

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53 represent single alleles ofhuman, murine and rat MASP-2 respectively, and that allelic variationand alternative splicing are expected to occur. Allelic variants of thenucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:50 and SEQ IDNO:53, including those containing silent mutations and those in whichmutations result in amino acid sequence changes, are within the scope ofthe present invention. Allelic variants of the MASP-2 sequence can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures.

The domains of the human MASP-2 protein (SEQ ID NO:6) are shown in FIGS.1 and 2A and include an N-terminal Clr/Cls/sea urchin Vegf/bonemorphogenic protein (CUBI) domain (aa 1-121 of SEQ ID NO:6), anepidermal growth factor-like domain (aa 122-166), a second CUBI domain(aa 167-293), as well as a tandem of complement control protein domainsand a serine protease domain. Alternative splicing of the MASP 2 generesults in MAp19 shown in FIG. 1. MAp19 is a nonenzymatic proteincontaining the N-terminal CUB 1-EGF region of MASP-2 with fouradditional residues (EQSL) derived from exon E as shown in FIG. 1.

Several proteins have been shown to bind to, or interact with MASP-2through protein-to-protein interactions. For example, MASP-2 is known tobind to, and form Ca²⁺ dependent complexes with, the lectin proteinsMBL, H-ficolin and L-ficolin. Each MASP-2/lectin complex has been shownto activate complement through the MASP-2-dependent cleavage of proteinsC4 and C2 (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987;Matsushita, M., et al., J. Exp. Med. 176:1497-2284, 2000; Matsushita,M., et al., J. Immunol. 168:3502-3506, 2002). Studies have shown thatthe CUB1-EGF domains of MASP-2 are essential for the association ofMASP-2 with MBL (Thielens, N. M., et al., J. Immunol. 166:5068, 2001).It has also been shown that the CUBIEGFCUBII domains mediatedimerization of MASP-2, which is required for formation of an active MBLcomplex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000).Therefore, MASP-2 inhibitory agents can be identified that bind to orinterfere with MASP-2 target regions known to be important forMASP-2-dependent complement activation.

Anti-MASP-2 Antibodies

In some embodiments of this aspect of the invention, the MASP-2inhibitory agent comprises an anti-MASP-2 antibody that inhibits theMASP-2-dependent complement activation system. The anti-MASP-2antibodies useful in this aspect of the invention include polyclonal,monoclonal or recombinant antibodies derived from any antibody producingmammal and may be multispecific, chimeric, humanized, anti-idiotype, andantibody fragments. Antibody fragments include Fab, Fab′, F(ab)₂,F(ab′)₂, Fv fragments, scFv fragments and single-chain antibodies asfurther described herein.

Several anti-MASP-2 antibodies have been described in the literature,some of which are listed below in TABLE 1. These previously describedanti-MASP-2 antibodies can be screened for the ability to inhibit theMASP-2-dependent complement activation system using the assays describedherein. For example, anti rat MASP-2 Fab2 antibodies have beenidentified that block MASP-2 dependent complement activation, asdescribed in more detail in Examples 10 and 11 herein. Once ananti-MASP-2 antibody is identified that functions as a MASP-2 inhibitoryagent, it can be used to produce anti-idiotype antibodies and used toidentify other MASP-2 binding molecules as further described below.

TABLE 1 MASP-2 SPECIFIC ANTIBODIES FROM THE LITERATURE ANTIGEN ANTIBODYTYPE REFERENCE Recombinant Rat Polyclonal Peterson, S.V., et al., Mol.MASP-2 Immunol. 37:803-811, 2000 Recombinant Rat MoAb Moller-Kristensen,M., et al., human (subclass IgG1) J. of Immunol. Methods CCP1/2-SP282:159-167, 2003 fragment (MoAb 8B5) Recombinant Rat MoAbMoller-Kristensen, M., et al., human (subclass IgG1) J. of Immunol.Methods MAp19 282:159-167, 2003 (MoAb 6G12) (cross reacts with MASP-2)hMASP-2 Mouse MoAb (S/P) Peterson, S.V., et al., Mol. Mouse MoAb(N-term) Immunol. 35:409, April 1998 hMASP-2 rat MoAb: Nimoab101, WO2004/106384 (CCP1- produced by hybridoma CCP2-SP cell line 03050904domain (ECACC) hMASP-2 murine MoAbs: WO 2004/106384 (full NimoAb104,produced length-his by hybridoma cell line tagged) M0545YM035 (DSMZ)NimoAb108, produced by hybridoma cell line M0545YM029 (DSMZ) NimoAb109produced by hybridoma cell line M0545YM046 (DSMZ) NimoAb110 produced byhybridoma cell line M0545YM048 (DSMZ)

Anti-MASP-2 Antibodies with Reduced Effector Function

In some embodiments of this aspect of the invention, the anti-MASP-2antibodies have reduced effector function in order to reduceinflammation that may arise from the activation of the classicalcomplement pathway. The ability of IgG molecules to trigger theclassical complement pathway has been shown to reside within the Fcportion of the molecule (Duncan, A. R., et al., Nature 332:738-7401988). IgG molecules in which the Fc portion of the molecule has beenremoved by enzymatic cleavage are devoid of this effector function (seeHarlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988). Accordingly, antibodies with reduced effector functioncan be generated as the result of lacking the Fc portion of the moleculeby having a genetically engineered Fc sequence that minimizes effectorfunction, or being of either the human IgG₂ or IgG4 isotype.

Antibodies with reduced effector function can be produced by standardmolecular biological manipulation of the Fc portion of the IgG heavychains as described in Example 9 herein and also described in Jolliffeet al., Int'l Rev. Immunol. 10:241-250, 1993, and Rodrigues et al., J.Immunol. 151:6954-6961, 1998. Antibodies with reduced effector functionalso include human IgG2 and IgG4 isotypes that have a reduced ability toactivate complement and/or interact with Fc receptors (Ravetch, J. V.,et al., Annu. Rev. Immunol. 9:457-492, 1991; Isaacs, J. D., et al., J.Immunol. 148:3062-3071, 1992; van de Winkel, J. G., et al., Immunol.Today 14:215-221, 1993). Humanized or fully human antibodies specific tohuman MASP-2 comprised of IgG2 or IgG4 isotypes can be produced by oneof several methods known to one of ordinary skilled in the art, asdescribed in Vaughan, T. J., et al., Nature Biotechnical 16:535-539,1998.

Production of Anti-MASP-2 Antibodies

Anti-MASP-2 antibodies can be produced using MASP-2 polypeptides (e.g.,full length MASP-2) or using antigenic MASP-2 epitope-bearing peptides(e.g., a portion of the MASP-2 polypeptide). Immunogenic peptides may beas small as five amino acid residues. For example, the MASP-2polypeptide including the entire amino acid sequence of SEQ ID NO:6 maybe used to induce anti-MASP-2 antibodies useful in the method of theinvention. Particular MASP-2 domains known to be involved inprotein-protein interactions, such as the CUBI, and CUBIEGF domains, aswell as the region encompassing the serine-protease active site, may beexpressed as recombinant polypeptides as described in Example 3 and usedas antigens. In addition, peptides comprising a portion of at least 6amino acids of the MASP-2 polypeptide (SEQ ID NO:6) are also useful toinduce MASP-2 antibodies. Additional examples of MASP-2 derived antigensuseful to induce MASP-2 antibodies are provided below in TABLE 2. TheMASP-2 peptides and polypeptides used to raise antibodies may beisolated as natural polypeptides, or recombinant or synthetic peptidesand catalytically inactive recombinant polypeptides, such as MASP-2A, asfurther described in Examples 5-7. In some embodiments of this aspect ofthe invention, anti-MASP-2 antibodies are obtained using a transgenicmouse strain as described in Examples 8 and 9 and further describedbelow.

Antigens useful for producing anti-MASP-2 antibodies also include fusionpolypeptides, such as fusions of MASP-2 or a portion thereof with animmunoglobulin polypeptide or with maltose-binding protein. Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is hapten-like, such portion may beadvantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

TABLE 2 MASP-2 DERIVED ANTIGENS SEQ ID NO: Amino Acid SequenceSEQ ID NO: 6 Human MASP-2 protein SEQ ID NO: 51 Murine MASP-2 proteinSEQ ID NO: 8 CUBI domain of human MASP-2 (aa 1-121 of SEQ ID NO: 6)SEQ ID NO: 9 CUBIEGF domains of human MASP-2 (aa 1-166 of SEQ ID NO: 6)SEQ ID NO: 10 CUBIEGFCUBII domains of human MASP-2(aa 1-293 of SEQ ID NO:6) SEQ ID NO: 11 EGF domain of human MASP-2(aa 122-166 of SEQ ID NO: 6) SEQ ID NO: 12 Serine-Protease domain ofhuman MASP-2 (aa 429-671 of SEQ ID NO: 6) SEQ ID NO: 13Serine-Protease inactivated GKDSCRGDAGGALVFL mutant form(aa 610-625 of SEQ ID NO: 6 with mutated Ser 618) SEQ ID NO: 14Human CUBI peptide TPLGPKWPEPVFGRL SEQ ID NO: 15: Human CUBI peptideTAPPGYRLRLYFTHFDLEL SHLCEYDFVKLSSGAKVL ATLCGQ SEQ ID NO: 16:MBL binding region in human TFRSDYSN CUBI domain SEQ ID NO: 17:MBL binding region in human FYSLGSSLDITFRSDYSNEK CUBI domain PFTGFSEQ ID NO: 18 EGF peptide IDECQVAPG SEQ ID NO: 19Peptide from serine-protease ANMLCAGLESGGKDSCRG active site DSGGALV

Polyclonal Antibodies

Polyclonal antibodies against MASP-2 can be prepared by immunizing ananimal with MASP-2 polypeptide or an immunogenic portion thereof usingmethods well known to those of ordinary skill in the art. See, forexample, Green et al., “Production of Polyclonal Antisera,” inImmunochemical Protocols (Manson, ed.), page 105, and as furtherdescribed in Example 6. The immunogenicity of a MASP-2 polypeptide canbe increased through the use of an adjuvant, including mineral gels,such as aluminum hydroxide or Freund's adjuvant (complete orincomplete), surface active substances such as lysolecithin, pluronicpolyols, polyanions, oil emulsions, keyhole limpet hemocyanin anddinitrophenol. Polyclonal antibodies are typically raised in animalssuch as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs,goats, or sheep. Alternatively, an anti-MASP-2 antibody useful in thepresent invention may also be derived from a subhuman primate. Generaltechniques for raising diagnostically and therapeutically usefulantibodies in baboons may be found, for example, in Goldenberg et al.,International Patent Publication No. WO 91/11465, and in Losman, M. J.,et al., Int. J. Cancer 46:310, 1990. Sera containing immunologicallyactive antibodies are then produced from the blood of such immunizedanimals using standard procedures well known in the art.

Monoclonal Antibodies

In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highlyspecific, being directed against a single MASP-2 epitope. As usedherein, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogenous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. Monoclonal antibodies can be obtainedusing any technique that provides for the production of antibodymolecules by continuous cell lines in culture, such as the hybridomamethod described by Kohler, G., et al., Nature 256:495, 1975, or theymay be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567 to Cabilly). Monoclonal antibodies may also be isolated fromphage antibody libraries using the techniques described in Clackson, T.,et al., Nature 352:624-628, 1991, and Marks, J. D., et al., J. Mol.Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

For example, monoclonal antibodies can be obtained by injecting asuitable mammal (e.g., a BALB/c mouse) with a composition comprising aMASP-2 polypeptide or portion thereof. After a predetermined period oftime, splenocytes are removed from the mouse and suspended in a cellculture medium. The splenocytes are then fused with an immortal cellline to form a hybridoma. The formed hybridomas are grown in cellculture and screened for their ability to produce a monoclonal antibodyagainst MASP-2. An example further describing the production ofanti-MASP-2 monoclonal antibodies is provided in Example 7. (See alsoCurrent Protocols in Immunology, Vol. 1., John Wiley & Sons, pages2.5.1-2.6.7, 1991.)

Human monoclonal antibodies may be obtained through the use oftransgenic mice that have been engineered to produce specific humanantibodies in response to antigenic challenge. In this technique,elements of the human immunoglobulin heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous immunoglobulin heavychain and light chain loci. The transgenic mice can synthesize humanantibodies specific for human antigens, such as the MASP-2 antigensdescribed herein, and the mice can be used to produce human MASP-2antibody-secreting hybridomas by fusing B-cells from such animals tosuitable myeloma cell lines using conventional Kohler-Milsteintechnology as further described in Example 7. Transgenic mice with ahuman immunoglobulin genome are commercially available (e.g., fromAbgenix, Inc., Fremont, Calif., and Medarex, Inc., Annandale, N.J.).Methods for obtaining human antibodies from transgenic mice aredescribed, for example, by Green, L. L., et al., Nature Genet. 7:13,1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, L. D., etal., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, The Humana Press, Inc., Vol. 10, pages 79-104, 1992).

Once produced, polyclonal, monoclonal or phage-derived antibodies arefirst tested for specific MASP-2 binding. A variety of assays known tothose skilled in the art may be utilized to detect antibodies whichspecifically bind to MASP-2. Exemplary assays include Western blot orimmunoprecipitation analysis by standard methods (e.g., as described inAusubel et al.), immunoelectrophoresis, enzyme-linked immuno-sorbentassays, dot blots, inhibition or competition assays and sandwich assays(as described in Harlow and Land, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1988). Once antibodies are identifiedthat specifically bind to MASP-2, the anti-MASP-2 antibodies are testedfor the ability to function as a MASP-2 inhibitory agent in one ofseveral assays such as, for example, a lectin-specific C4 cleavage assay(described in Example 2), a C3b deposition assay (described in Example2) or a C4b deposition assay (described in Example 2).

The affinity of anti-MASP-2 monoclonal antibodies can be readilydetermined by one of ordinary skill in the art (see, e.g., Scatchard,A., NY Acad. Sci. 51:660-672, 1949). In one embodiment, the anti-MASP-2monoclonal antibodies useful for the methods of the invention bind toMASP-2 with a binding affinity of <100 nM, preferably <10 nM and mostpreferably <2 nM. In some embodiments, a MASP-2 inhibitory monoclonalantibody useful in the methods of the invention is a MASP-2 inhibitorymonoclonal antibody, or antigen binding fragment thereof, comprising (I)(a) a heavy-chain variable region comprising: i) a heavy-chain CDR-H1comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) aheavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acidsequence from 95-102 of SEQ ID NO:67 and b) a light-chain variableregion comprising: i) a light-chain CDR-L1 comprising the amino acidsequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii)a light-chain CDR-L3 comprising the amino acid sequence from 89-97 ofSEQ ID NO:70, or (II) a variant thereof comprising a heavy-chainvariable region with at least 90% identity to SEQ ID NO:67 (e.g., atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to SEQ IDNO:67) and a light-chain variable region with at least 90% identity(e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% identity toSEQ ID NO:70.

Chimeric/Humanized Antibodies

Monoclonal antibodies useful in the method of the invention includechimeric antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies (U.S. Pat. No. 4,816,567, toCabilly; and Morrison, S. L., et al., Proc. Nat'l Acad. Sci. USA81:6851-6855, 1984).

One form of a chimeric antibody useful in the invention is a humanizedmonoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g.,murine) antibodies are chimeric antibodies, which contain minimalsequence derived from non-human immunoglobulin. Humanized monoclonalantibodies are produced by transferring the non-human (e.g., mouse)complementarity determining regions (CDR), from the heavy and lightvariable chains of the mouse immunoglobulin into a human variabledomain. Typically, residues of human antibodies are then substituted inthe framework regions of the non-human counterparts. Furthermore,humanized antibodies may comprise residues that are not found in therecipient antibody or in the donor antibody. These modifications aremade to further refine antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo variable domains, in which all or substantially all of thehypervariable loops correspond to those of a non-human immunoglobulinand all or substantially all of the Fv framework regions are those of ahuman immunoglobulin sequence. The humanized antibody optionally alsowill comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones, P. T., et al., Nature 321:522-525, 1986; Reichmann, L., et al.,Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596,1992.

The humanized antibodies useful in the invention include humanmonoclonal antibodies including at least a MASP-2 binding CDR3 region.In addition, the Fc portions may be replaced so as to produce IgA or IgMas well as human IgG antibodies. Such humanized antibodies will haveparticular clinical utility because they will specifically recognizehuman MASP-2 but will not evoke an immune response in humans against theantibody itself. Consequently, they are better suited for in vivoadministration in humans, especially when repeated or long-termadministration is necessary.

An example of the generation of a humanized anti-MASP-2 antibody from amurine anti-MASP-2 monoclonal antibody is provided herein in Example 6.Techniques for producing humanized monoclonal antibodies are alsodescribed, for example, by Jones, P. T., et al., Nature 321:522, 1986;Carter, P., et al., Proc. Nat'l. Acad. Sci. USA 89:4285, 1992; Sandhu,J. S., Crit. Rev. Biotech. 12:437, 1992; Singer, I. I., et al., J.Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols,Humana Press, Inc., 1995; Kelley, “Engineering Therapeutic Antibodies,”in Protein Engineering: Principles and Practice, Cleland et al. (eds.),John Wiley & Sons, Inc., pages 399-434, 1996; and by U.S. Pat. No.5,693,762, to Queen, 1997. In addition, there are commercial entitiesthat will synthesize humanized antibodies from specific murine antibodyregions, such as Protein Design Labs (Mountain View, Calif.).

Recombinant Antibodies

Anti-MASP-2 antibodies can also be made using recombinant methods. Forexample, human antibodies can be made using human immunoglobulinexpression libraries (available for example, from Stratagene, Corp., LaJolla, Calif.) to produce fragments of human antibodies (V_(H), V_(L),Fv, Fd, Fab or F(ab′)₂). These fragments are then used to constructwhole human antibodies using techniques similar to those for producingchimeric antibodies.

Anti-Idiotype Antibodies

Once anti-MASP-2 antibodies are identified with the desired inhibitoryactivity, these antibodies can be used to generate anti-idiotypeantibodies that resemble a portion of MASP-2 using techniques that arewell known in the art. See, e.g., Greenspan, N. S., et al., FASEB J.7:437, 1993. For example, antibodies that bind to MASP-2 andcompetitively inhibit a MASP-2 protein interaction required forcomplement activation can be used to generate anti-idiotypes thatresemble the MBL binding site on MASP-2 protein and therefore bind andneutralize a binding ligand of MASP-2 such as, for example, MBL.

Immunoglobulin Fragments

The MASP-2 inhibitory agents useful in the method of the inventionencompass not only intact immunoglobulin molecules but also the wellknown fragments including Fab, Fab′, F(ab)₂, F(ab′)₂ and Fv fragments,scFv fragments, diabodies, linear antibodies, single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

It is well known in the art that only a small portion of an antibodymolecule, the paratope, is involved in the binding of the antibody toits epitope (see, e.g., Clark, W. R., The Experimental Foundations ofModern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc′ and Fcregions of the antibody are effectors of the classical complementpathway, but are not involved in antigen binding. An antibody from whichthe pFc′ region has been enzymatically cleaved, or which has beenproduced without the pFc′ region, is designated an F(ab′)₂ fragment andretains both of the antigen binding sites of an intact antibody. Anisolated F(ab′)₂ fragment is referred to as a bivalent monoclonalfragment because of its two antigen binding sites. Similarly, anantibody from which the Fc region has been enzymatically cleaved, orwhich has been produced without the Fc region, is designated a Fabfragment, and retains one of the antigen binding sites of an intactantibody molecule.

Antibody fragments can be obtained by proteolytic hydrolysis, such as bypepsin or papain digestion of whole antibodies by conventional methods.For example, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent to produce3.5S Fab′ monovalent fragments. Optionally, the cleavage reaction can beperformed using a blocking group for the sulfhydryl groups that resultfrom cleavage of disulfide linkages. As an alternative, an enzymaticcleavage using pepsin produces two monovalent Fab fragments and an Fcfragment directly. These methods are described, for example, U.S. Pat.No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem.Biophys. 89:230, 1960; Porter, R. R., Biochem. J. 73:119, 1959; Edelman,et al., in Methods in Enzymology 1:422, Academic Press, 1967; and byColigan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

In some embodiments, the use of antibody fragments lacking the Fc regionare preferred to avoid activation of the classical complement pathwaywhich is initiated upon binding Fc to the Fcγ receptor. There areseveral methods by which one can produce a MoAb that avoids Fcγ receptorinteractions. For example, the Fc region of a monoclonal antibody can beremoved chemically using partial digestion by proteolytic enzymes (suchas ficin digestion), thereby generating, for example, antigen-bindingantibody fragments such as Fab or F(ab)₂ fragments (Mariani, M., et al.,Mol. Immunol. 28:69-71, 1991). Alternatively, the human γ4 IgG isotype,which does not bind Fcγ receptors, can be used during construction of ahumanized antibody as described herein. Antibodies, single chainantibodies and antigen-binding domains that lack the Fc domain can alsobe engineered using recombinant techniques described herein.

Single-Chain Antibody Fragments

Alternatively, one can create single peptide chain binding moleculesspecific for MASP-2 in which the heavy and light chain Fv regions areconnected. The Fv fragments may be connected by a peptide linker to forma single-chain antigen binding protein (scFv). These single-chainantigen binding proteins are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains which areconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are described for example, by Whitlow, etal., “Methods: A Companion to Methods in Enzymology” 2:97, 1991; Bird,et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778, to Ladner; Pack,P., et al., Bio/Technology 11:1271, 1993.

As an illustrative example, a MASP-2 specific scFv can be obtained byexposing lymphocytes to MASP-2 polypeptide in vitro and selectingantibody display libraries in phage or similar vectors (for example,through the use of immobilized or labeled MASP-2 protein or peptide).Genes encoding polypeptides having potential MASP-2 polypeptide bindingdomains can be obtained by screening random peptide libraries displayedon phage or on bacteria such as E. coli. These random peptide displaylibraries can be used to screen for peptides which interact with MASP-2.Techniques for creating and screening such random peptide displaylibraries are well known in the art (U.S. Pat. No. 5,223,409, toLardner; U.S. Pat. No. 4,946,778, to Ladner; U.S. Pat. No. 5,403,484, toLardner; U.S. Pat. No. 5,571,698, to Lardner; and Kay et al., PhageDisplay of Peptides and Proteins Academic Press, Inc., 1996) and randompeptide display libraries and kits for screening such libraries areavailable commercially, for instance from CLONTECH Laboratories, Inc.(Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New EnglandBiolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc.(Piscataway, N.J.).

Another form of an anti-MASP-2 antibody fragment useful in this aspectof the invention is a peptide coding for a singlecomplementarity-determining region (CDR) that binds to an epitope on aMASP-2 antigen and inhibits MASP-2-dependent complement activation. CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells(see, for example, Larrick et al., Methods: A Companion to Methods inEnzymology 2:106, 1991; Courtenay-Luck, “Genetic Manipulation ofMonoclonal Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), page 166,Cambridge University Press, 1995; and Ward et al., “Genetic Manipulationand Expression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, Birch et al. (eds.), page 137, Wiley-Liss, Inc., 1995).

The MASP-2 antibodies described herein are administered to a subject inneed thereof to inhibit MASP-2-dependent complement activation. In someembodiments, the MASP-2 inhibitory agent is a high-affinity human orhumanized monoclonal anti-MASP-2 antibody with reduced effectorfunction.

Peptide Inhibitors

In some embodiments of this aspect of the invention, the MASP-2inhibitory agent comprises isolated MASP-2 peptide inhibitors, includingisolated natural peptide inhibitors and synthetic peptide inhibitorsthat inhibit the MASP-2-dependent complement activation system. As usedherein, the term “isolated MASP-2 peptide inhibitors” refers to peptidesthat inhibit MASP-2 dependent complement activation by binding to,competing with MASP-2 for binding to another recognition molecule (e.g.,MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, and/ordirectly interacting with MASP-2 to inhibit MASP-2-dependent complementactivation that are substantially pure and are essentially free of othersubstances with which they may be found in nature to an extent practicaland appropriate for their intended use.

Peptide inhibitors have been used successfully in vivo to interfere withprotein-protein interactions and catalytic sites. For example, peptideinhibitors to adhesion molecules structurally related to LFA-1 haverecently been approved for clinical use in coagulopathies (Ohman, E. M.,et al., European Heart J. 16:50-55, 1995). Short linear peptides (<30amino acids) have been described that prevent or interfere withintegrin-dependent adhesion (Murayama, O., et al., J. Biochem.120:445-51, 1996). Longer peptides, ranging in length from 25 to 200amino acid residues, have also been used successfully to blockintegrin-dependent adhesion (Zhang, L., et al., J. Biol. Chem.271(47):29953-57, 1996). In general, longer peptide inhibitors havehigher affinities and/or slower off-rates than short peptides and maytherefore be more potent inhibitors. Cyclic peptide inhibitors have alsobeen shown to be effective inhibitors of integrins in vivo for thetreatment of human inflammatory disease (Jackson, D. Y., et al., J. Med.Chem. 40:3359-68, 1997). One method of producing cyclic peptidesinvolves the synthesis of peptides in which the terminal amino acids ofthe peptide are cysteines, thereby allowing the peptide to exist in acyclic form by disulfide bonding between the terminal amino acids, whichhas been shown to improve affinity and half-life in vivo for thetreatment of hematopoietic neoplasms (e.g., U.S. Pat. No. 6,649,592, toLarson).

Synthetic MASP-2 Peptide Inhibitors

MASP-2 inhibitory peptides useful in the methods of this aspect of theinvention are exemplified by amino acid sequences that mimic the targetregions important for MASP-2 function. The inhibitory peptides useful inthe practice of the methods of the invention range in size from about 5amino acids to about 300 amino acids. TABLE 3 provides a list ofexemplary inhibitory peptides that may be useful in the practice of thisaspect of the present invention. A candidate MASP-2 inhibitory peptidemay be tested for the ability to function as a MASP-2 inhibitory agentin one of several assays including, for example, a lectin specific C4cleavage assay (described in Example 2), and a C3b deposition assay(described in Example 2).

In some embodiments, the MASP-2 inhibitory peptides are derived fromMASP-2 polypeptides and are selected from the full length mature MASP-2protein (SEQ ID NO:6), or from a particular domain of the MASP-2 proteinsuch as, for example, the CUBI domain (SEQ ID NO:8), the CUBIEGF domain(SEQ ID NO:9), the EGF domain (SEQ ID NO:11), and the serine proteasedomain (SEQ ID NO:12). As previously described, the CUBEGFCUBII regionshave been shown to be required for dimerization and binding with MBL(Thielens et al., supra). In particular, the peptide sequence TFRSDYN(SEQ ID NO:16) in the CUBI domain of MASP-2 has been shown to beinvolved in binding to MBL in a study that identified a human carrying ahomozygous mutation at Asp105 to Gly105, resulting in the loss of MASP-2from the MBL complex (Stengaard-Pedersen, K., et al., New England J.Med. 349:554-560, 2003).

In some embodiments, MASP-2 inhibitory peptides are derived from thelectin proteins that bind to MASP-2 and are involved in the lectincomplement pathway. Several different lectins have been identified thatare involved in this pathway, including mannan-binding lectin (MBL),L-ficolin, M-ficolin and H-ficolin. (Ikeda, K., et al., J. Biol. Chem.262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. 176:1497-2284,2000; Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). Theselectins are present in serum as oligomers of homotrimeric subunits, eachhaving N-terminal collagen-like fibers with carbohydrate recognitiondomains. These different lectins have been shown to bind to MASP-2, andthe lectin/MASP-2 complex activates complement through cleavage ofproteins C4 and C2. H-ficolin has an amino-terminal region of 24 aminoacids, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domainof 12 amino acids, and a fibrinogen-like domain of 207 amino acids(Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). H-ficolinbinds to GIcNAc and agglutinates human erythrocytes coated with LPSderived from S. typhimurium, S. minnesota and E. coli. H-ficolin hasbeen shown to be associated with MASP-2 and MAp19 and activates thelectin pathway. Id. L-ficolin/P35 also binds to GIcNAc and has beenshown to be associated with MASP-2 and MAp19 in human serum and thiscomplex has been shown to activate the lectin pathway (Matsushita, M.,et al., J. Immunol. 164:2281, 2000). Accordingly, MASP-2 inhibitorypeptides useful in the present invention may comprise a region of atleast 5 amino acids selected from the MBL protein (SEQ ID NO:21), theH-ficolin protein (Genbank accession number NM_173452), the M-ficolinprotein (Genbank accession number 000602) and the L-ficolin protein(Genbank accession number NM_015838).

More specifically, scientists have identified the MASP-2 binding site onMBL to be within the 12 Gly-X-Y triplets “GKD GRD GTK GEK GEP GQG LRGLQG POG KLG POG NOG PSG SOG PKG QKG DOG KS” (SEQ ID NO:26) that liebetween the hinge and the neck in the C-terminal portion of thecollagen-like domain of MBP (Wallis, R., et al., J. Biol. Chem.279:14065, 2004). This MASP-2 binding site region is also highlyconserved in human H-ficolin and human L-ficolin. A consensus bindingsite has been described that is present in all three lectin proteinscomprising the amino acid sequence “OGK-X-GP” (SEQ ID NO:22) where theletter “O” represents hydroxyproline and the letter “X” is a hydrophobicresidue (Wallis et al., 2004, supra). Accordingly, in some embodiments,MASP-2 inhibitory peptides useful in this aspect of the invention are atleast 6 amino acids in length and comprise SEQ ID NO:22. Peptidesderived from MBL that include the amino acid sequence “GLR GLQ GPO GKLGPO G” (SEQ ID NO:24) have been shown to bind MASP-2 in vitro (Wallis,et al., 2004, supra). To enhance binding to MASP-2, peptides can besynthesized that are flanked by two GPO triplets at each end (“GPO GPOGLR GLQ GPO GKL GPO GGP OGP O” SEQ ID NO:25) to enhance the formation oftriple helices as found in the native MBL protein (as further describedin Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).

MASP-2 inhibitory peptides may also be derived from human H-ficolin thatinclude the sequence “GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO” (SEQID NO:27) from the consensus MASP-2 binding region in H-ficolin. Alsoincluded are peptides derived from human L-ficolin that include thesequence “GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAO GEO”(SEQ ID NO:28) from the consensus MASP-2 binding region in L-ficolin.

MASP-2 inhibitory peptides may also be derived from the C4 cleavage sitesuch as “LQRALEILPNRVTIKANRPFLVFI” (SEQ ID NO:29) which is the C4cleavage site linked to the C-terminal portion of antithrombin III(Glover, G. I., et al., Mol. Immunol. 25:1261 (1988)).

TABLE 3 EXEMPLARY MASP-2 INHIBITORY PEPTIDES SEQ ID NO SourceSEQ ID NO: 6 Human MASP-2 protein SEQ ID NO: 8 CUBI domain of MASP-2(aa 1-121 of SEQ ID NO: 6) SEQ ID NO: 9 CUBIEGF domains of MASP-2(aa 1-166 of SEQ ID NO: 6) SEQ ID NO: 10 CUBIEGFCUBII domains ofMASP-2 (aa 1-293 of  SEQ ID NO: 6) SEQ ID NO: 11 EGF domain of MASP-2(aa 122-166) SEQ ID NO: 12 Serine-protease domain of MASP-2 (aa 429-671)SEQ ID NO: 16 MBL binding region in MASP-2 SEQ ID NO: 3 Human MAp19SEQ ID NO: 21 Human MBL protein SEQ ID NO: 22Synthetic peptide Consensus OGK-X-GP, binding site from HumanWhere ″O″ = MBL and Human ficolins hydroxyproline and ″X″is a hydrophobic amino acid residue SEQ ID NO: 23Human MBL core binding site OGKLG SEQ ID NO: 24 Human MBP Triplets 6-10-GLR GLQ GPO GKL demonstrated binding to GPO G MASP-2 SEQ ID NO: 25Human MBP Triplets with GPO GPOGPOGLRGLQGPO added to enhance formationGKLGPOGGPOGPO of triple helices SEQ ID NO: 26 Human MBP Triplets 1-17GKDGRDGTKGEKGEP GQGLRGLQGPOGKLG POGNOGPSGSOGPKG QKGDOGKS SEQ ID NO: 27Human H-Ficolin (Hataka) GAOGSOGEKGAOGPQ GPOGPOGKMGPKGEO GDOSEQ ID NO: 28 Human L-Ficolin P35 GCOGLOGAOGDKGE AGTNGKRGERGPOGPOGKAGPOGPNGAOGE O SEQ ID NO: 29 Human C4 cleavage site LQRALEILPNRVTIKANRPFLVFI Note: The letter ″O″ represents hydroxyproline. The letter″X″ is a hydrophobic residue.

Peptides derived from the C4 cleavage site as well as other peptidesthat inhibit the MASP-2 serine protease site can be chemically modifiedso that they are irreversible protease inhibitors. For example,appropriate modifications may include, but are not necessarily limitedto, halomethyl ketones (Br, Cl, I, F) at the C-terminus, Asp or Glu, orappended to functional side chains; haloacetyl (or other α-haloacetyl)groups on amino groups or other functional side chains; epoxide orimine-containing groups on the amino or carboxy termini or on functionalside chains; or imidate esters on the amino or carboxy termini or onfunctional side chains. Such modifications would afford the advantage ofpermanently inhibiting the enzyme by covalent attachment of the peptide.This could result in lower effective doses and/or the need for lessfrequent administration of the peptide inhibitor.

In addition to the inhibitory peptides described above, MASP-2inhibitory peptides useful in the method of the invention includepeptides containing the MASP-2-binding CDR3 region of anti-MASP-2 MoAbobtained as described herein. The sequence of the CDR regions for use insynthesizing the peptides may be determined by methods known in the art.The heavy chain variable region is a peptide that generally ranges from100 to 150 amino acids in length. The light chain variable region is apeptide that generally ranges from 80 to 130 amino acids in length. TheCDR sequences within the heavy and light chain variable regions includeonly approximately 3-25 amino acid sequences that may be easilysequenced by one of ordinary skill in the art.

Those skilled in the art will recognize that substantially homologousvariations of the MASP-2 inhibitory peptides described above will alsoexhibit MASP-2 inhibitory activity. Exemplary variations include, butare not necessarily limited to, peptides having insertions, deletions,replacements, and/or additional amino acids on the carboxy-terminus oramino-terminus portions of the subject peptides and mixtures thereof.Accordingly, those homologous peptides having MASP-2 inhibitory activityare considered to be useful in the methods of this invention. Thepeptides described may also include duplicating motifs and othermodifications with conservative substitutions. Conservative variants aredescribed elsewhere herein, and include the exchange of an amino acidfor another of like charge, size or hydrophobicity and the like.

MASP-2 inhibitory peptides may be modified to increase solubility and/orto maximize the positive or negative charge in order to more closelyresemble the segment in the intact protein. The derivative may or maynot have the exact primary amino acid structure of a peptide disclosedherein so long as the derivative functionally retains the desiredproperty of MASP-2 inhibition. The modifications can include amino acidsubstitution with one of the commonly known twenty amino acids or withanother amino acid, with a derivatized or substituted amino acid withancillary desirable characteristics, such as resistance to enzymaticdegradation or with a D-amino acid or substitution with another moleculeor compound, such as a carbohydrate, which mimics the naturalconfirmation and function of the amino acid, amino acids or peptide;amino acid deletion; amino acid insertion with one of the commonly knowntwenty amino acids or with another amino acid, with a derivatized orsubstituted amino acid with ancillary desirable characteristics, such asresistance to enzymatic degradation or with a D-amino acid orsubstitution with another molecule or compound, such as a carbohydrate,which mimics the natural confirmation and function of the amino acid,amino acids or peptide; or substitution with another molecule orcompound, such as a carbohydrate or nucleic acid monomer, which mimicsthe natural conformation, charge distribution and function of the parentpeptide. Peptides may also be modified by acetylation or amidation.

The synthesis of derivative inhibitory peptides can rely on knowntechniques of peptide biosynthesis, carbohydrate biosynthesis and thelike. As a starting point, the artisan may rely on a suitable computerprogram to determine the conformation of a peptide of interest. Once theconformation of peptide disclosed herein is known, then the artisan candetermine in a rational design fashion what sort of substitutions can bemade at one or more sites to fashion a derivative that retains the basicconformation and charge distribution of the parent peptide but which maypossess characteristics which are not present or are enhanced over thosefound in the parent peptide. Once candidate derivative molecules areidentified, the derivatives can be tested to determine if they functionas MASP-2 inhibitory agents using the assays described herein.

Screening for MASP-2 Inhibitory Peptides

One may also use molecular modeling and rational molecular design togenerate and screen for peptides that mimic the molecular structures ofkey binding regions of MASP-2 and inhibit the complement activities ofMASP-2. The molecular structures used for modeling include the CDRregions of anti-MASP-2 monoclonal antibodies, as well as the targetregions known to be important for MASP-2 function including the regionrequired for dimerization, the region involved in binding to MBL, andthe serine protease active site as previously described. Methods foridentifying peptides that bind to a particular target are well known inthe art. For example, molecular imprinting may be used for the de novoconstruction of macromolecular structures such as peptides that bind toa particular molecule. See, for example, Shea, K. J., “MolecularImprinting of Synthetic Network Polymers: The De Novo synthesis ofMacromolecular Binding and Catalytic Sties,” TRIP 2(5) 1994.

As an illustrative example, one method of preparing mimics of MASP-2binding peptides is as follows. Functional monomers of a known MASP-2binding peptide or the binding region of an anti-MASP-2 antibody thatexhibits MASP-2 inhibition (the template) are polymerized. The templateis then removed, followed by polymerization of a second class ofmonomers in the void left by the template, to provide a new moleculethat exhibits one or more desired properties that are similar to thetemplate. In addition to preparing peptides in this manner, other MASP-2binding molecules that are MASP-2 inhibitory agents such aspolysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins,carbohydrates, glycoproteins, steroid, lipids and other biologicallyactive materials can also be prepared. This method is useful fordesigning a wide variety of biological mimics that are more stable thantheir natural counterparts because they are typically prepared by freeradical polymerization of function monomers, resulting in a compoundwith a nonbiodegradable backbone.

Peptide Synthesis

The MASP-2 inhibitory peptides can be prepared using techniques wellknown in the art, such as the solid-phase synthetic technique initiallydescribed by Merrifield, in J. Amer. Chem. Soc. 85:2149-2154, 1963.Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordancewith the instructions provided by the manufacturer. Other techniques maybe found, for example, in Bodanszky, M., et al., Peptide Synthesis,second edition, John Wiley & Sons, 1976, as well as in other referenceworks known to those skilled in the art.

The peptides can also be prepared using standard genetic engineeringtechniques known to those skilled in the art. For example, the peptidecan be produced enzymatically by inserting nucleic acid encoding thepeptide into an expression vector, expressing the DNA, and translatingthe DNA into the peptide in the presence of the required amino acids.The peptide is then purified using chromatographic or electrophoretictechniques, or by means of a carrier protein that can be fused to, andsubsequently cleaved from, the peptide by inserting into the expressionvector in phase with the peptide encoding sequence a nucleic acidsequence encoding the carrier protein. The fusion protein-peptide may beisolated using chromatographic, electrophoretic or immunologicaltechniques (such as binding to a resin via an antibody to the carrierprotein). The peptide can be cleaved using chemical methodology orenzymatically, as by, for example, hydrolases.

The MASP-2 inhibitory peptides that are useful in the method of theinvention can also be produced in recombinant host cells followingconventional techniques. To express a MASP-2 inhibitory peptide encodingsequence, a nucleic acid molecule encoding the peptide must be operablylinked to regulatory sequences that control transcriptional expressionin an expression vector and then introduced into a host cell. Inaddition to transcriptional regulatory sequences, such as promoters andenhancers, expression vectors can include translational regulatorysequences and a marker gene, which are suitable for selection of cellsthat carry the expression vector.

Nucleic acid molecules that encode a MASP-2 inhibitory peptide can besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically synthesized double-stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes, synthetic genes (double-stranded) are assembled in modularform from single-stranded fragments that are from 20 to 100 nucleotidesin length. For reviews on polynucleotide synthesis, see, for example,Glick and Pasternak, “Molecular Biotechnology, Principles andApplications of Recombinant DNA”, ASM Press, 1994; Itakura, K., et al.,Annu. Rev. Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nat'lAcad. Sci. USA 87:633, 1990.

Small Molecule Inhibitors

In some embodiments, MASP-2 inhibitory agents are small moleculeinhibitors including natural and synthetic substances that have a lowmolecular weight, such as for example, peptides, peptidomimetics andnonpeptide inhibitors (including oligonucleotides and organiccompounds). Small molecule inhibitors of MASP-2 can be generated basedon the molecular structure of the variable regions of the anti-MASP-2antibodies.

Small molecule inhibitors may also be designed and generated based onthe MASP-2 crystal structure using computational drug design (Kuntz I.D., et al., Science 257:1078, 1992). The crystal structure of rat MASP-2has been described (Feinberg, H., et al., EMBO J. 22:2348-2359, 2003).Using the method described by Kuntz et al., the MASP-2 crystal structurecoordinates are used as an input for a computer program such as DOCK,which outputs a list of small molecule structures that are expected tobind to MASP-2. Use of such computer programs is well known to one ofskill in the art. For example, the crystal structure of the HIV-1protease inhibitor was used to identify unique nonpeptide ligands thatare HIV-1 protease inhibitors by evaluating the fit of compounds foundin the Cambridge Crystallographic database to the binding site of theenzyme using the program DOCK (Kuntz, I. D., et al., J. Mol. Biol.161:269-288, 1982; DesJarlais, R. L., et al., PNAS 87:6644-6648, 1990).

The list of small molecule structures that are identified by acomputational method as potential MASP-2 inhibitors are screened using aMASP-2 binding assay such as described in Example 10. The smallmolecules that are found to bind to MASP-2 are then assayed in afunctional assay such as described in Example 2 to determine if theyinhibit MASP-2-dependent complement activation.

MASP-2 Soluble Receptors

Other suitable MASP-2 inhibitory agents are believed to include MASP-2soluble receptors, which may be produced using techniques known to thoseof ordinary skill in the art.

Expression Inhibitors of MASP-2

In another embodiment of this aspect of the invention, the MASP-2inhibitory agent is a MASP-2 expression inhibitor capable of inhibitingMASP-2-dependent complement activation. In the practice of this aspectof the invention, representative MASP-2 expression inhibitors includeMASP-2 antisense nucleic acid molecules (such as antisense mRNA,antisense DNA or antisense oligonucleotides), MASP-2 ribozymes andMASP-2 RNAi molecules.

Anti-sense RNA and DNA molecules act to directly block the translationof MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing translationof MASP-2 protein. An antisense nucleic acid molecule may be constructedin a number of different ways provided that it is capable of interferingwith the expression of MASP-2. For example, an antisense nucleic acidmolecule can be constructed by inverting the coding region (or a portionthereof) of MASP-2 cDNA (SEQ ID NO:4) relative to its normal orientationfor transcription to allow for the transcription of its complement.

The antisense nucleic acid molecule is usually substantially identicalto at least a portion of the target gene or genes. The nucleic acid,however, need not be perfectly identical to inhibit expression.Generally, higher homology can be used to compensate for the use of ashorter antisense nucleic acid molecule. The minimal percent identity istypically greater than about 65%, but a higher percent identity mayexert a more effective repression of expression of the endogenoussequence. Substantially greater percent identity of more than about 80%typically is preferred, though about 95% to absolute identity istypically most preferred.

The antisense nucleic acid molecule need not have the same intron orexon pattern as the target gene, and non-coding segments of the targetgene may be equally effective in achieving antisense suppression oftarget gene expression as coding segments. A DNA sequence of at leastabout 8 or so nucleotides may be used as the antisense nucleic acidmolecule, although a longer sequence is preferable. In the presentinvention, a representative example of a useful inhibitory agent ofMASP-2 is an antisense MASP-2 nucleic acid molecule which is at leastninety percent identical to the complement of the MASP-2 cDNA consistingof the nucleic acid sequence set forth in SEQ ID NO:4. The nucleic acidsequence set forth in SEQ ID NO:4 encodes the MASP-2 protein consistingof the amino acid sequence set forth in SEQ ID NO:5.

The targeting of antisense oligonucleotides to bind MASP-2 mRNA isanother mechanism that may be used to reduce the level of MASP-2 proteinsynthesis. For example, the synthesis of polygalacturonase and themuscarine type 2 acetylcholine receptor is inhibited by antisenseoligonucleotides directed to their respective mRNA sequences (U.S. Pat.No. 5,739,119, to Cheng, and U.S. Pat. No. 5,759,829, to Shewmaker).Furthermore, examples of antisense inhibition have been demonstratedwith the nuclear protein cyclin, the multiple drug resistance gene(MDG1), ICAM-1, E-selectin, STK-1, striatal GABA_(A) receptor and humanEGF (see, e.g., U.S. Pat. No. 5,801,154, to Baracchini; U.S. Pat. No.5,789,573, to Baker; U.S. Pat. No. 5,718,709, to Considine; and U.S.Pat. No. 5,610,288, to Reubenstein).

A system has been described that allows one of ordinary skill todetermine which oligonucleotides are useful in the invention, whichinvolves probing for suitable sites in the target mRNA using Rnase Hcleavage as an indicator for accessibility of sequences within thetranscripts. Scherr, M., et al., Nucleic Acids Res. 26:5079-5085, 1998;Lloyd, et al., Nucleic Acids Res. 29:3665-3673, 2001. A mixture ofantisense oligonucleotides that are complementary to certain regions ofthe MASP-2 transcript is added to cell extracts expressing MASP-2, suchas hepatocytes, and hybridized in order to create an RNAseH vulnerablesite. This method can be combined with computer-assisted sequenceselection that can predict optimal sequence selection for antisensecompositions based upon their relative ability to form dimers, hairpins,or other secondary structures that would reduce or prohibit specificbinding to the target mRNA in a host cell. These secondary structureanalysis and target site selection considerations may be performed usingthe OLIGO primer analysis software (Rychlik, I., 1997) and the BLASTN2.0.5 algorithm software (Altschul, S. F., et al., Nucl. Acids Res.25:3389-3402, 1997). The antisense compounds directed towards the targetsequence preferably comprise from about 8 to about 50 nucleotides inlength. Antisense oligonucleotides comprising from about 9 to about 35or so nucleotides are particularly preferred. The inventors contemplateall oligonucleotide compositions in the range of 9 to 35 nucleotides(i.e., those of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or so bases inlength) are highly preferred for the practice of antisenseoligonucleotide-based methods of the invention. Highly preferred targetregions of the MASP-2 mRNA are those that are at or near the AUGtranslation initiation codon, and those sequences that are substantiallycomplementary to 5′ regions of the mRNA, e.g., between the −10 and +10regions of the MASP-2 gene nucleotide sequence (SEQ ID NO:4). ExemplaryMASP-2 expression inhibitors are provided in TABLE 4.

TABLE 4 EXEMPLARY EXPRESSION INHIBITORS OF MASP-2SEQ ID NO: 30 (nucleotides Nucleic acid sequence 22-680 of SEQ ID NO: 4)of MASP-2 cDNA (SEQ ID NO: 4 encoding CUBIEGF SEQ ID NO: 31Nucleotides 12-45 of 5′CGGGCACACCATGAGGCTGCTG SEQ ID NO: 4 ACCCTCCTGGGC3including the MASP-2 translation start site (sense) SEQ ID NO: 32Nucleotides 361-396 of 5′GACATTACCTTCCGCTCCGACTC SEQ ID NO: 4CAACGAGAAG3′ encoding a region comprising the MASP-2 MBL binding site(sense) SEQ ID NO: 33 Nucleotides 610-642 of 5′AGCAGCCCTGAATACCCACGGCCSEQ ID NO: 4 GTATCCCAAA3′ encoding a region comprising the CUBII domain

As noted above, the term “oligonucleotide” as used herein refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics thereof. This term also covers those oligonucleobasescomposed of naturally occurring nucleotides, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally occurring modifications. These modifications allow one tointroduce certain desirable properties that are not offered throughnaturally occurring oligonucleotides, such as reduced toxic properties,increased stability against nuclease degradation and enhanced cellularuptake. In illustrative embodiments, the antisense compounds of theinvention differ from native DNA by the modification of thephosphodiester backbone to extend the life of the antisenseoligonucleotide in which the phosphate substituents are replaced byphosphorothioates. Likewise, one or both ends of the oligonucleotide maybe substituted by one or more acridine derivatives that intercalatebetween adjacent basepairs within a strand of nucleic acid.

Another alternative to antisense is the use of “RNA interference”(RNAi). Double-stranded RNAs (dsRNAs) can provoke gene silencing inmammals in vivo. The natural function of RNAi and co-suppression appearsto be protection of the genome against invasion by mobile geneticelements such as retrotransposons and viruses that produce aberrant RNAor dsRNA in the host cell when they become active (see, e.g., Jensen,J., et al., Nat. Genet. 21:209-12, 1999). The double-stranded RNAmolecule may be prepared by synthesizing two RNA strands capable offorming a double-stranded RNA molecule, each having a length from about19 to 25 (e.g., 19-23 nucleotides). For example, a dsRNA molecule usefulin the methods of the invention may comprise the RNA corresponding to asequence and its complement listed in TABLE 4. Preferably, at least onestrand of RNA has a 3′ overhang from 1-5 nucleotides. The synthesizedRNA strands are combined under conditions that form a double-strandedmolecule. The RNA sequence may comprise at least an 8 nucleotide portionof SEQ ID NO:4 with a total length of 25 nucleotides or less. The designof siRNA sequences for a given target is within the ordinary skill ofone in the art. Commercial services are available that design siRNAsequence and guarantee at least 70% knockdown of expression (Qiagen,Valencia, Calif.).

The dsRNA may be administered as a pharmaceutical composition andcarried out by known methods, wherein a nucleic acid is introduced intoa desired target cell. Commonly used gene transfer methods includecalcium phosphate, DEAE-dextran, electroporation, microinjection andviral methods. Such methods are taught in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

Ribozymes can also be utilized to decrease the amount and/or biologicalactivity of MASP-2, such as ribozymes that target MASP-2 mRNA. Ribozymesare catalytic RNA molecules that can cleave nucleic acid moleculeshaving a sequence that is completely or partially homologous to thesequence of the ribozyme. It is possible to design ribozyme transgenesthat encode RNA ribozymes that specifically pair with a target RNA andcleave the phosphodiester backbone at a specific location, therebyfunctionally inactivating the target RNA. In carrying out this cleavage,the ribozyme is not itself altered, and is thus capable of recycling andcleaving other molecules. The inclusion of ribozyme sequences withinantisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the antisense constructs.

Ribozymes useful in the practice of the invention typically comprise ahybridizing region of at least about nine nucleotides, which iscomplementary in nucleotide sequence to at least part of the targetMASP-2 mRNA, and a catalytic region that is adapted to cleave the targetMASP-2 mRNA (see generally, EPA No. 0 321 201; WO88/04300; Haseloff, J.,et al., Nature 334:585-591, 1988; Fedor, M. J., et al., Proc. Natl.Acad. Sci. USA 87:1668-1672, 1990; Cech, T. R., et al., Ann. Rev.Biochem. 55:599-629, 1986).

Ribozymes can either be targeted directly to cells in the form of RNAoligonucleotides incorporating ribozyme sequences, or introduced intothe cell as an expression vector encoding the desired ribozymal RNA.Ribozymes may be used and applied in much the same way as described forantisense polynucleotides.

Anti-sense RNA and DNA, ribozymes and RNAi molecules useful in themethods of the invention may be prepared by any method known in the artfor the synthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art, such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well known modifications of the DNA molecules may be introducedas a means of increasing stability and half-life. Useful modificationsinclude, but are not limited to, the addition of flanking sequences ofribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

VI. PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS

Dosing

In another aspect, the invention provides compositions for inhibitingthe adverse effects of MASP-2-dependent complement activation in asubject suffering from a disease or condition as disclosed herein,comprising administering to the subject a composition comprising atherapeutically effective amount of a MASP-2 inhibitory agent and apharmaceutically acceptable carrier. The MASP-2 inhibitory agents can beadministered to a subject in need thereof, at therapeutically effectivedoses to treat or ameliorate conditions associated with MASP-2-dependentcomplement activation. A therapeutically effective dose refers to theamount of the MASP-2 inhibitory agent sufficient to result inamelioration of symptoms associated with the disease or condition.

Toxicity and therapeutic efficacy of MASP-2 inhibitory agents can bedetermined by standard pharmaceutical procedures employing experimentalanimal models, such as the murine MASP-2 −/− mouse model expressing thehuman MASP-2 transgene described in Example 1. Using such animal models,the NOAEL (no observed adverse effect level) and the MED (the minimallyeffective dose) can be determined using standard methods. The dose ratiobetween NOAEL and MED effects is the therapeutic ratio, which isexpressed as the ratio NOAEL/MED. MASP-2 inhibitory agents that exhibitlarge therapeutic ratios or indices are most preferred. The dataobtained from the cell culture assays and animal studies can be used informulating a range of dosages for use in humans. The dosage of theMASP-2 inhibitory agent preferably lies within a range of circulatingconcentrations that include the MED with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

For any compound formulation, the therapeutically effective dose can beestimated using animal models. For example, a dose may be formulated inan animal model to achieve a circulating plasma concentration range thatincludes the MED. Quantitative levels of the MASP-2 inhibitory agent inplasma may also be measured, for example, by high performance liquidchromatography.

In addition to toxicity studies, effective dosage may also be estimatedbased on the amount of MASP-2 protein present in a living subject andthe binding affinity of the MASP-2 inhibitory agent. It has been shownthat MASP-2 levels in normal human subjects is present in serum in lowlevels in the range of 500 ng/ml, and MASP-2 levels in a particularsubject can be determined using a quantitative assay for MASP-2described in Moller-Kristensen M., et al., J. Immunol. Methods282:159-167, 2003.

Generally, the dosage of administered compositions comprising MASP-2inhibitory agents varies depending on such factors as the subject's age,weight, height, sex, general medical condition, and previous medicalhistory. As an illustration, MASP-2 inhibitory agents, such asanti-MASP-2 antibodies, can be administered in dosage ranges from about0.010 to 10.0 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably0.010 to 0.1 mg/kg of the subject body weight. In some embodiments thecomposition comprises a combination of anti-MASP-2 antibodies and MASP-2inhibitory peptides.

Therapeutic efficacy of MASP-2 inhibitory compositions and methods ofthe present invention in a given subject, and appropriate dosages, canbe determined in accordance with complement assays well known to thoseof skill in the art. Complement generates numerous specific products.During the last decade, sensitive and specific assays have beendeveloped and are available commercially for most of these activationproducts, including the small activation fragments C3a, C4a, and C5a andthe large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of theseassays utilize monoclonal antibodies that react with new antigens(neoantigens) exposed on the fragment, but not on the native proteinsfrom which they are formed, making these assays very simple andspecific. Most rely on ELISA technology, although radioimmunoassay isstill sometimes used for C3a and C5a. These latter assays measure boththe unprocessed fragments and their ‘desArg’ fragments, which are themajor forms found in the circulation. Unprocessed fragments andC5a_(desArg) are rapidly cleared by binding to cell surface receptorsand are hence present in very low concentrations, whereas C3a_(desArg)does not bind to cells and accumulates in plasma. Measurement of C3aprovides a sensitive, pathway-independent indicator of complementactivation. Alternative pathway activation can be assessed by measuringthe Bb fragment. Detection of the fluid-phase product of membrane attackpathway activation, sC5b-9, provides evidence that complement is beingactivated to completion. Because both the lectin and classical pathwaysgenerate the same activation products, C4a and C4d, measurement of thesetwo fragments does not provide any information about which of these twopathways has generated the activation products.

The inhibition of MASP-2-dependent complement activation ischaracterized by at least one of the following changes in a component ofthe complement system that occurs as a result of administration of aMASP-2 inhibitory agent in accordance with the methods of the invention:the inhibition of the generation or production of MASP-2-dependentcomplement activation system products C4b, C3a, C5a and/or C5b-9 (MAC)(measured, for example, as described in measured, for example, asdescribed in Example 2, the reduction of C4 cleavage and C4b deposition(measured, for example as described in Example 10), or the reduction ofC3 cleavage and C3b deposition (measured, for example, as described inExample 10).

Additional Agents

The compositions and methods comprising MASP-2 inhibitory agents mayoptionally comprise one or more additional therapeutic agents, which mayaugment the activity of the MASP-2 inhibitory agent or that providerelated therapeutic functions in an additive or synergistic fashion. Forexample, in the context of treating a subject suffering from TTP,wherein the subject is positive for an inhibitor of ADAM-TS13, one ormore MASP-2 inhibitory agents may be administered in combination(including co-administration) with one or more immunosuppressive agents.Suitable immunosuppressive agents include: corticosteroids, rituxan,cyclosporine, and the like. In the context of treating a subjectsuffering from, or at risk for developing, HUS or aHUS, one or moreMASP-2 inhibitory agents may be administered in combination (includingco-administration) with a suitable antibiotic. In the context oftreating a subject suffering from, or at risk for developing aHUS, oneor more MASP-2 inhibitory agents may be administered in combination(including co-administration) with other complement inhibitory agentssuch as eculizumab (Soliris), TT-30, antibody to factor B, or otheragents that inhibit terminal complement components or alternativepathway amplification.

The inclusion and selection of additional agent(s) will be determined toachieve a desired therapeutic result. In some embodiments, the MASP-2inhibitory agent may be administered in combination with one or moreanti-inflammatory and/or analgesic agents. Suitable anti-inflammatoryand/or analgesic agents include: serotonin receptor antagonists;serotonin receptor agonists; histamine receptor antagonists; bradykininreceptor antagonists; kallikrein inhibitors; tachykinin receptorantagonists, including neurokinin₁ and neurokinin₂ receptor subtypeantagonists; calcitonin gene-related peptide (CGRP) receptorantagonists; interleukin receptor antagonists; inhibitors of enzymesactive in the synthetic pathway for arachidonic acid metabolites,including phospholipase inhibitors, including PLA₂ isoform inhibitorsand PLC_(γ) isoform inhibitors, cyclooxygenase (COX) inhibitors (whichmay be either COX-1, COX-2, or nonselective COX-1 and -2 inhibitors),lipooxygenase inhibitors; prostanoid receptor antagonists includingeicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxanereceptor subtype antagonists; leukotriene receptor antagonists includingleukotriene B₄ receptor subtype antagonists and leukotriene D₄ receptorsubtype antagonists; opioid receptor agonists, including μ-opioid,δ-opioid, and κ-opioid receptor subtype agonists; purinoceptor agonistsand antagonists including P_(2X) receptor antagonists and P_(2Y)receptor agonists; adenosine triphosphate (ATP)-sensitive potassiumchannel openers; MAP kinase inhibitors; nicotinic acetylcholineinhibitors; and alpha adrenergic receptor agonists (including alpha-1,alpha-2, and nonselective alpha-1 and 2 agonists).

The MASP-2 inhibitory agents of the present invention may also beadministered in combination with one or more other complementinhibitors, such as an inhibitor of C5. To date, Eculizumab (Solaris®),an antibody against C5, is the only complement-targeting drug that hasbeen approved for human use. However some pharmacological agents havebeen shown to block complement in vivo. K76COOH and nafamstat mesilateare two agents that have shown some effectiveness in animal models oftransplantation (Miyagawa, S., et al., Transplant Proc. 24:483-484,1992). Low molecular weight heparins have also been shown to beeffective in regulating complement activity (Edens, R. E., et al.,Complement Today, pp. 96-120, Basel: Karger, 1993). It is believed thatthese small molecule inhibitors may be useful as agents to use incombination with the MASP-2 inhibitory agents of the present invention.

Other naturally occurring complement inhibitors may be useful incombination with the MASP-2 inhibitory agents of the present invention.Biological inhibitors of complement include soluble complement factor 1(sCRI). This is a naturally-occurring inhibitor that can be found on theouter membrane of human cells. Other membrane inhibitors include DAF,MCP, and CD59. Recombinant forms have been tested for theiranti-complement activity in vitro and in vivo. sCR1 has been shown to beeffective in xenotransplantation, wherein the complement system (bothalternative and classical) provides the trigger for a hyperactiverejection syndrome within minutes of perfusing blood through the newlytransplanted organ (Platt, J. L., et al., Immunol. Today 11:450-6, 1990;Marino, I. R., et al., Transplant Proc. 1071:6, 1990; Johnstone, P. S.,et al., Transplantation 54:573-6, 1992). The use of sCR1 protects andextends the survival time of the transplanted organ, implicating thecomplement pathway in the pathogenesis of organ survival (Leventhal, J.R., et al., Transplantation 55:857-66, 1993; Pruitt, S. K., et al.,Transplantation 57:363-70, 1994).

Suitable additional complement inhibitors for use in combination withthe compositions of the present invention also include, by way ofexample, MoAbs such as an anti-C5 antibody (e.g., eculizumab) beingdeveloped by Alexion Pharmaceuticals, Inc., New Haven, Conn., andanti-properdin MoAbs.

Pharmaceutical Carriers and Delivery Vehicles

In general, the MASP-2 inhibitory agent compositions of the presentinvention, combined with any other selected therapeutic agents, aresuitably contained in a pharmaceutically acceptable carrier. The carrieris non-toxic, biocompatible and is selected so as not to detrimentallyaffect the biological activity of the MASP-2 inhibitory agent (and anyother therapeutic agents combined therewith). Exemplary pharmaceuticallyacceptable carriers for peptides are described in U.S. Pat. No.5,211,657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptidesuseful in the invention may be formulated into preparations in solid,semi-solid, gel, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, depositories, inhalants andinjections allowing for oral, parenteral or surgical administration. Theinvention also contemplates local administration of the compositions bycoating medical devices and the like.

Suitable carriers for parenteral delivery via injectable, infusion orirrigation and topical delivery include distilled water, physiologicalphosphate-buffered saline, normal or lactated Ringer's solutions,dextrose solution, Hank's solution, or propanediol. In addition,sterile, fixed oils may be employed as a solvent or suspending medium.For this purpose any biocompatible oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables. The carrier and agentmay be compounded as a liquid, suspension, polymerizable ornon-polymerizable gel, paste or salve.

The carrier may also comprise a delivery vehicle to sustain (i.e.,extend, delay or regulate) the delivery of the agent(s) or to enhancethe delivery, uptake, stability or pharmacokinetics of the therapeuticagent(s). Such a delivery vehicle may include, by way of non-limitingexample, microparticles, microspheres, nanospheres or nanoparticlescomposed of proteins, liposomes, carbohydrates, synthetic organiccompounds, inorganic compounds, polymeric or copolymeric hydrogels andpolymeric micelles. Suitable hydrogel and micelle delivery systemsinclude the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexesdisclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrincomplexes disclosed in U.S. Patent Application Publication No.2002/0019369 A1. Such hydrogels may be injected locally at the site ofintended action, or subcutaneously or intramuscularly to form asustained release depot.

For intra-articular delivery, the MASP-2 inhibitory agent may be carriedin above-described liquid or gel carriers that are injectable,above-described sustained-release delivery vehicles that are injectable,or a hyaluronic acid or hyaluronic acid derivative.

For oral administration of non-peptidergic agents, the MASP-2 inhibitoryagent may be carried in an inert filler or diluent such as sucrose,cornstarch, or cellulose.

For topical administration, the MASP-2 inhibitory agent may be carriedin ointment, lotion, cream, gel, drop, suppository, spray, liquid orpowder, or in gel or microcapsular delivery systems via a transdermalpatch.

Various nasal and pulmonary delivery systems, including aerosols,metered-dose inhalers, dry powder inhalers, and nebulizers, are beingdeveloped and may suitably be adapted for delivery of the presentinvention in an aerosol, inhalant, or nebulized delivery vehicle,respectively.

For intrathecal (IT) or intracerebroventricular (ICV) delivery,appropriately sterile delivery systems (e.g., liquids; gels,suspensions, etc.) can be used to administer the present invention.

The compositions of the present invention may also include biocompatibleexcipients, such as dispersing or wetting agents, suspending agents,diluents, buffers, penetration enhancers, emulsifiers, binders,thickeners, flavouring agents (for oral administration).

Pharmaceutical Carriers for Antibodies and Peptides

More specifically with respect to anti-MASP-2 antibodies and inhibitorypeptides, exemplary formulations can be parenterally administered asinjectable dosages of a solution or suspension of the compound in aphysiologically acceptable diluent with a pharmaceutical carrier thatcan be a sterile liquid such as water, oils, saline, glycerol orethanol. Additionally, auxiliary substances such as wetting oremulsifying agents, surfactants, pH buffering substances and the likecan be present in compositions comprising anti-MASP-2 antibodies andinhibitory peptides. Additional components of pharmaceuticalcompositions include petroleum (such as of animal, vegetable orsynthetic origin), for example, soybean oil and mineral oil. In general,glycols such as propylene glycol or polyethylene glycol are preferredliquid carriers for injectable solutions.

The anti-MASP-2 antibodies and inhibitory peptides can also beadministered in the form of a depot injection or implant preparationthat can be formulated in such a manner as to permit a sustained orpulsatile release of the active agents.

Pharmaceutically Acceptable Carriers for Expression

Inhibitors

More specifically with respect to expression inhibitors useful in themethods of the invention, compositions are provided that comprise anexpression inhibitor as described above and a pharmaceuticallyacceptable carrier or diluent. The composition may further comprise acolloidal dispersion system.

Pharmaceutical compositions that include expression inhibitors mayinclude, but are not limited to, solutions, emulsions, andliposome-containing formulations. These compositions may be generatedfrom a variety of components that include, but are not limited to,preformed liquids, self-emulsifying solids and self-emulsifyingsemisolids. The preparation of such compositions typically involvescombining the expression inhibitor with one or more of the following:buffers, antioxidants, low molecular weight polypeptides, proteins,amino acids, carbohydrates including glucose, sucrose or dextrins,chelating agents such as EDTA, glutathione and other stabilizers andexcipients. Neutral buffered saline or saline mixed with non-specificserum albumin are examples of suitable diluents.

In some embodiments, the compositions may be prepared and formulated asemulsions which are typically heterogeneous systems of one liquiddispersed in another in the form of droplets (see, Idson, inPharmaceutical Dosage Forms, Vol. 1, Rieger and Banker (eds.), MarcekDekker, Inc., N.Y., 1988). Examples of naturally occurring emulsifiersused in emulsion formulations include acacia, beeswax, lanolin, lecithinand phosphatides.

In one embodiment, compositions including nucleic acids can beformulated as microemulsions. A microemulsion, as used herein refers toa system of water, oil, and amphiphile, which is a single opticallyisotropic and thermodynamically stable liquid solution (see Rosoff inPharmaceutical Dosage Forms, Vol. 1). The method of the invention mayalso use liposomes for the transfer and delivery of antisenseoligonucleotides to the desired site.

Pharmaceutical compositions and formulations of expression inhibitorsfor topical administration may include transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids andpowders. Conventional pharmaceutical carriers, as well as aqueous,powder or oily bases and thickeners and the like may be used.

Modes of Administration

The pharmaceutical compositions comprising MASP-2 inhibitory agents maybe administered in a number of ways depending on whether a local orsystemic mode of administration is most appropriate for the conditionbeing treated. Additionally, as described herein above with respect toextracorporeal reperfusion procedures, MASP-2 inhibitory agents can beadministered via introduction of the compositions of the presentinvention to recirculating blood or plasma. Further, the compositions ofthe present invention can be delivered by coating or incorporating thecompositions on or into an implantable medical device.

Systemic Delivery

As used herein, the terms “systemic delivery” and “systemicadministration” are intended to include but are not limited to oral andparenteral routes including intramuscular (IM), subcutaneous,intravenous (IV), intra-arterial, inhalational, sublingual, buccal,topical, transdermal, nasal, rectal, vaginal and other routes ofadministration that effectively result in dispersement of the deliveredagent to a single or multiple sites of intended therapeutic action.Preferred routes of systemic delivery for the present compositionsinclude intravenous, intramuscular, subcutaneous and inhalational. Itwill be appreciated that the exact systemic administration route forselected agents utilized in particular compositions of the presentinvention will be determined in part to account for the agent'ssusceptibility to metabolic transformation pathways associated with agiven route of administration. For example, peptidergic agents may bemost suitably administered by routes other than oral.

MASP-2 inhibitory antibodies and polypeptides can be delivered into asubject in need thereof by any suitable means. Methods of delivery ofMASP-2 antibodies and polypeptides include administration by oral,pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous(IV) or subcutaneous injection), inhalation (such as via a fine powderformulation), transdermal, nasal, vaginal, rectal, or sublingual routesof administration, and can be formulated in dosage forms appropriate foreach route of administration.

By way of representative example, MASP-2 inhibitory antibodies andpeptides can be introduced into a living body by application to a bodilymembrane capable of absorbing the polypeptides, for example the nasal,gastrointestinal and rectal membranes. The polypeptides are typicallyapplied to the absorptive membrane in conjunction with a permeationenhancer. (See, e.g., Lee, V. H. L., Crit. Rev. Ther. Drug Carrier Sys.5:69, 1988; Lee, V. H. L., J. Controlled Release 13:213, 1990; Lee, V.H. L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York(1991); DeBoer, A. G., et al., J. Controlled Release 13:241, 1990.) Forexample, STDHF is a synthetic derivative of fusidic acid, a steroidalsurfactant that is similar in structure to the bile salts, and has beenused as a permeation enhancer for nasal delivery. (Lee, W. A., Biopharm.22, November/December 1990.) The MASP-2 inhibitory antibodies andpolypeptides may be introduced in association with another molecule,such as a lipid, to protect the polypeptides from enzymatic degradation.For example, the covalent attachment of polymers, especiallypolyethylene glycol (PEG), has been used to protect certain proteinsfrom enzymatic hydrolysis in the body and thus prolong half-life(Fuertges, F., et al., J. Controlled Release 11:139, 1990). Many polymersystems have been reported for protein delivery (Bae, Y. H., et al., J.Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813,1989; Yamakawa, I., et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I.,et al., J. Controlled Release 10:195, 1989; Asano, M., et al., J.Controlled Release 9:111, 1989; Rosenblatt, J., et al., J. ControlledRelease 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990;Takakura, Y., et al., J. Pharm. Sci. 78:117, 1989; Takakura, Y., et al.,J. Pharm. Sci. 78:219, 1989).

Recently, liposomes have been developed with improved serum stabilityand circulation half-times (see, e.g., U.S. Pat. No. 5,741,516, toWebb). Furthermore, various methods of liposome and liposome-likepreparations as potential drug carriers have been reviewed (see, e.g.,U.S. Pat. No. 5,567,434, to Szoka; U.S. Pat. No. 5,552,157, to Yagi;U.S. Pat. No. 5,565,213, to Nakamori; U.S. Pat. No. 5,738,868, toShinkarenko; and U.S. Pat. No. 5,795,587, to Gao).

For transdermal applications, the MASP-2 inhibitory antibodies andpolypeptides may be combined with other suitable ingredients, such ascarriers and/or adjuvants. There are no limitations on the nature ofsuch other ingredients, except that they must be pharmaceuticallyacceptable for their intended administration, and cannot degrade theactivity of the active ingredients of the composition. Examples ofsuitable vehicles include ointments, creams, gels, or suspensions, withor without purified collagen. The MASP-2 inhibitory antibodies andpolypeptides may also be impregnated into transdermal patches, plasters,and bandages, preferably in liquid or semi-liquid form.

The compositions of the present invention may be systemicallyadministered on a periodic basis at intervals determined to maintain adesired level of therapeutic effect. For example, compositions may beadministered, such as by subcutaneous injection, every two to four weeksor at less frequent intervals. The dosage regimen will be determined bythe physician considering various factors that may influence the actionof the combination of agents. These factors will include the extent ofprogress of the condition being treated, the patient's age, sex andweight, and other clinical factors. The dosage for each individual agentwill vary as a function of the MASP-2 inhibitory agent that is includedin the composition, as well as the presence and nature of any drugdelivery vehicle (e.g., a sustained release delivery vehicle). Inaddition, the dosage quantity may be adjusted to account for variationin the frequency of administration and the pharmacokinetic behavior ofthe delivered agent(s).

Local Delivery

As used herein, the term “local” encompasses application of a drug in oraround a site of intended localized action, and may include for exampletopical delivery to the skin or other affected tissues, ophthalmicdelivery, intrathecal (IT), intracerebroventricular (ICV),intra-articular, intracavity, intracranial or intravesicularadministration, placement or irrigation. Local administration may bepreferred to enable administration of a lower dose, to avoid systemicside effects, and for more accurate control of the timing of deliveryand concentration of the active agents at the site of local delivery.Local administration provides a known concentration at the target site,regardless of interpatient variability in metabolism, blood flow, etc.Improved dosage control is also provided by the direct mode of delivery.

Local delivery of a MASP-2 inhibitory agent may be achieved in thecontext of surgical methods for treating a disease or condition, such asfor example during procedures such as arterial bypass surgery,atherectomy, laser procedures, ultrasonic procedures, balloonangioplasty and stent placement. For example, a MASP-2 inhibitor can beadministered to a subject in conjunction with a balloon angioplastyprocedure. A balloon angioplasty procedure involves inserting a catheterhaving a deflated balloon into an artery. The deflated balloon ispositioned in proximity to the atherosclerotic plaque and is inflatedsuch that the plaque is compressed against the vascular wall. As aresult, the balloon surface is in contact with the layer of vascularendothelial cells on the surface of the blood vessel. The MASP-2inhibitory agent may be attached to the balloon angioplasty catheter ina manner that permits release of the agent at the site of theatherosclerotic plaque. The agent may be attached to the ballooncatheter in accordance with standard procedures known in the art. Forexample, the agent may be stored in a compartment of the ballooncatheter until the balloon is inflated, at which point it is releasedinto the local environment. Alternatively, the agent may be impregnatedon the balloon surface, such that it contacts the cells of the arterialwall as the balloon is inflated. The agent may also be delivered in aperforated balloon catheter such as those disclosed in Flugelman, M. Y.,et al., Circulation 85:1110-1117, 1992. See also published PCTApplication WO 95/23161 for an exemplary procedure for attaching atherapeutic protein to a balloon angioplasty catheter. Likewise, theMASP-2 inhibitory agent may be included in a gel or polymeric coatingapplied to a stent, or may be incorporated into the material of thestent, such that the stent elutes the MASP-2 inhibitory agent aftervascular placement.

MASP-2 inhibitory compositions used in the treatment of arthritides andother musculoskeletal disorders may be locally delivered byintra-articular injection. Such compositions may suitably include asustained release delivery vehicle. As a further example of instances inwhich local delivery may be desired, MASP-2 inhibitory compositions usedin the treatment of urogenital conditions may be suitably instilledintravesically or within another urogenital structure.

Coatings on a Medical Device

MASP-2 inhibitory agents such as antibodies and inhibitory peptides maybe immobilized onto (or within) a surface of an implantable orattachable medical device. The modified surface will typically be incontact with living tissue after implantation into an animal body. By“implantable or attachable medical device” is intended any device thatis implanted into, or attached to, tissue of an animal body, during thenormal operation of the device (e.g., stents and implantable drugdelivery devices). Such implantable or attachable medical devices can bemade from, for example, nitrocellulose, diazocellulose, glass,polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran,Sepharose, agar, starch, nylon, stainless steel, titanium andbiodegradable and/or biocompatible polymers. Linkage of the protein to adevice can be accomplished by any technique that does not destroy thebiological activity of the linked protein, for example by attaching oneor both of the N- C-terminal residues of the protein to the device.Attachment may also be made at one or more internal sites in theprotein. Multiple attachments (both internal and at the ends of theprotein) may also be used. A surface of an implantable or attachablemedical device can be modified to include functional groups (e.g.,carboxyl, amide, amino, ether, hydroxyl, cyano, nitrido, sulfanamido,acetylinic, epoxide, silanic, anhydric, succinimic, azido) for proteinimmobilization thereto. Coupling chemistries include, but are notlimited to, the formation of esters, ethers, amides, azido andsulfanamido derivatives, cyanate and other linkages to the functionalgroups available on MASP-2 antibodies or inhibitory peptides. MASP-2antibodies or inhibitory fragments can also be attached non-covalentlyby the addition of an affinity tag sequence to the protein, such as GST(D. B. Smith and K. S. Johnson, Gene 67:31, 1988), polyhistidines (E.Hochuli et al., J. Chromatog. 411:77, 1987), or biotin. Such affinitytags may be used for the reversible attachment of the protein to adevice.

Proteins can also be covalently attached to the surface of a devicebody, for example, by covalent activation of the surface of the medicaldevice. By way of representative example, matricellular protein(s) canbe attached to the device body by any of the following pairs of reactivegroups (one member of the pair being present on the surface of thedevice body, and the other member of the pair being present on thematricellular protein(s)): hydroxyl/carboxylic acid to yield an esterlinkage; hydroxyl/anhydride to yield an ester linkage;hydroxyl/isocyanate to yield a urethane linkage. A surface of a devicebody that does not possess useful reactive groups can be treated withradio-frequency discharge plasma (RFGD) etching to generate reactivegroups in order to allow deposition of matricellular protein(s) (e.g.,treatment with oxygen plasma to introduce oxygen-containing groups;treatment with propyl amino plasma to introduce amine groups).

MASP-2 inhibitory agents comprising nucleic acid molecules such asantisense, RNAi- or DNA-encoding peptide inhibitors can be embedded inporous matrices attached to a device body. Representative porousmatrices useful for making the surface layer are those prepared fromtendon or dermal collagen, as may be obtained from a variety ofcommercial sources (e.g., Sigma and Collagen Corporation), or collagenmatrices prepared as described in U.S. Pat. No. 4,394,370, to Jefferies,and U.S. Pat. No. 4,975,527, to Koezuka. One collagenous material istermed UltraFiber™ and is obtainable from Norian Corp. (Mountain View,Calif.).

Certain polymeric matrices may also be employed if desired, and includeacrylic ester polymers and lactic acid polymers, as disclosed, forexample, in U.S. Pat. Nos. 4,526,909 and 4,563,489, to Urist. Particularexamples of useful polymers are those of orthoesters, anhydrides,propylene-cofumarates, or a polymer of one or more a-hydroxy carboxylicacid monomers, (e.g., a-hydroxy acetic acid (glycolic acid) and/ora-hydroxy propionic acid (lactic acid)).

Treatment Regimens

In prophylactic applications, the pharmaceutical compositions areadministered to a subject susceptible to, or otherwise at risk of, acondition associated with MASP-2-dependent complement activation in anamount sufficient to eliminate or reduce the risk of developing symptomsof the condition. In therapeutic applications, the pharmaceuticalcompositions are administered to a subject suspected of, or alreadysuffering from, a condition associated with MASP-2-dependent complementactivation in a therapeutically effective amount sufficient to relieve,or at least partially reduce, the symptoms of the condition. In bothprophylactic and therapeutic regimens, compositions comprising MASP-2inhibitory agents may be administered in several dosages until asufficient therapeutic outcome has been achieved in the subject.Application of the MASP-2 inhibitory compositions of the presentinvention may be carried out by a single administration of thecomposition, or a limited sequence of administrations, for treatment ofan acute condition, e.g., reperfusion injury or other traumatic injury.Alternatively, the composition may be administered at periodic intervalsover an extended period of time for treatment of chronic conditions,e.g., arthritides or psoriasis.

The methods and compositions of the present invention may be used toinhibit inflammation and related processes that typically result fromdiagnostic and therapeutic medical and surgical procedures. To inhibitsuch processes, the MASP-2 inhibitory composition of the presentinvention may be applied periprocedurally. As used herein“periprocedurally” refers to administration of the inhibitorycomposition preprocedurally and/or intraprocedurally and/orpostprocedurally, i.e., before the procedure, before and during theprocedure, before and after the procedure, before, during and after theprocedure, during the procedure, during and after the procedure, orafter the procedure. Periprocedural application may be carried out bylocal administration of the composition to the surgical or proceduralsite, such as by injection or continuous or intermittent irrigation ofthe site or by systemic administration. Suitable methods for localperioperative delivery of MASP-2 inhibitory agent solutions aredisclosed in U.S. Pat. No. 6,420,432 to Demopulos and U.S. Pat. No.6,645,168 to Demopulos. Suitable methods for local delivery ofchondroprotective compositions including MASP-2 inhibitory agent(s) aredisclosed in International PCT Patent Application WO 01/07067 A2.Suitable methods and compositions for targeted systemic delivery ofchondroprotective compositions including MASP-2 inhibitory agent(s) aredisclosed in International PCT Patent Application WO 03/063799 A2.

In one aspect of the invention, the pharmaceutical compositions areadministered to a subject suffering from, or at risk for developing athrombotic microangiopathy (TMA). In one embodiment, the TMA is selectedfrom the group consisting of hemolytic uremic syndrome (HUS), thromboticthrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome(aHUS). In one embodiment, the TMA is aHUS. In one embodiment, thecomposition is administered to an aHUS patient during the acute phase ofthe disease. In one embodiment, the composition is administered to anaHUS patient during the remission phase (i.e., in a subject that hasrecovered or partially recovered from an episode of acute phase aHUS,such remission evidenced, for example, by increased platelet countand/or reduced serum LDH concentrations, for example as described inLoirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, herebyincorporated herein by reference). In one embodiment, the subject issuffering from, or at risk for developing a TMA that is (i) a TMAsecondary to cancer; (ii) a TMA secondary to chemotherapy; or (iii) aTMA secondary to transplantation (e.g., organ transplantation, such askidney transplantation or allogeneic hematopoietic stem celltransplantation). In one embodiment, the subject is suffering from, orat risk for developing Upshaw-Schulman Syndrome (USS). In oneembodiment, the subject is suffering from, or at risk for developingDegos disease. In one embodiment, the subject is suffering from, or atrisk for developing Catastrophic Antiphospholipid Syndrome (CAPS). Intherapeutic applications, the pharmaceutical compositions areadministered to a subject suffering from, or at risk for developing aTMA in a therapeutically effective amount sufficient to inhibit thrombusformation, relieve, or at least partially reduce, the symptoms of thecondition.

In both prophylactic and therapeutic regimens, compositions comprisingMASP-2 inhibitory agents may be administered in several dosages until asufficient therapeutic outcome has been achieved in the subject. In oneembodiment of the invention, the MASP-2 inhibitory agent comprises ananti-MASP-2 antibody, which suitably may be administered to an adultpatient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still moresuitably from 50.0 mg to 500 mg. For pediatric patients, dosage can beadjusted in proportion to the patient's weight. Application of theMASP-2 inhibitory compositions of the present invention may be carriedout by a single administration of the composition, or a limited sequenceof administrations, for treatment of TMA. Alternatively, the compositionmay be administered at periodic intervals such as daily, biweekly,weekly, every other week, monthly or bimonthly over an extended periodof time for treatment of TMA.

In some embodiments, the subject suffering from or at risk fordeveloping a TMA has previously undergone, or is currently undergoingtreatment with a terminal complement inhibitor that inhibits cleavage ofcomplement protein C5. In some embodiments, the method comprisesadministering to the subject a composition of the invention comprising aMASP-2 inhibitor and further administering to the subject a terminalcomplement inhibitor that inhibits cleavage of complement protein C5. Insome embodiments, the terminal complement inhibitor is a humanizedanti-C5 antibody or antigen-binding fragment thereof. In someembodiments, the terminal complement inhibitor is eculizumab.

In one aspect of the invention, the pharmaceutical compositions areadministered to a subject susceptible to, or otherwise at risk of, aHUSin an amount sufficient to eliminate or reduce the risk of developingsymptoms of the condition. In therapeutic applications, thepharmaceutical compositions are administered to a subject suspected of,or already suffering from, aHUS in a therapeutically effective amountsufficient to relieve, or at least partially reduce, the symptoms of thecondition. In one aspect of the invention, prior to administration, thesubject may be examined to determine whether the subject exhibits one ormore symptoms of aHUS, including (i) anemia, (ii) thrombocytopenia (iii)renal insufficiency and (iv) rising creatinine, and the composition ofthe present invention is then administered in an effective amount andfor a sufficient time period to improve these symptom(s).

In another aspect of the invention, the MASP-2 inhibitory compositionsof the present invention may be used to prophylactically treat a subjectthat has an elevated risk of developing aHUS and thereby reduce thelikelihood that the subject will deliver aHUS. The presence of a geneticmarker in the subject known to be associated with aHUS is firstdetermined by performing a genetic screening test on a sample obtainedfrom the subject and identifying the presence of at least one geneticmarker associated with aHUS, complement factor H (CFH), factor I (CFI),factor B (CFB), membrane cofactor CD46, C3, complement factor H-relatedprotein (CFHR1), anticoagulant protein thrombodulin (THBD), complementfactor H-related protein 3 (CFHR3) or complement factor H-relatedprotein 4 (CFHR4). The subject is then periodically monitored (e.g.,monthly, quarterly, twice annually or annually) to determine thepresence or absence of at least one symptom of aHUS, such as anemia,thrombocytopenia, renal insufficiency and rising creatinine. Upon thedetermination of the presence of at least one of these symptoms, thesubject can be administered an amount of a MASP-2 inhibitory agenteffective to inhibit MASP-2 dependent complement activation, in aneffective amount and for a sufficient time period to improve said one ormore symptoms.

In a still further aspect of the present invention, a subject atincreased risk of developing aHUS due to having been screened anddetermined to have one of the genetic markers associated with aHUS maybe monitored for the occurrence of an event associated with triggeringaHUS clinical symptoms, including drug exposure, infection (e.g.,bacterial infection), malignancy, injury, organ or tissue transplant andpregnancy.

In a still further aspect of the present invention, a compositioncomprising an amount of a MASP-2 inhibitory agent effective to inhibitMASP-2 dependent complement activation can be administered to asuffering from or at risk of developing atypical hemolytic uremicsyndrome (aHUS) secondary to an infection. For example, a patientsuffering from or at risk of developing non-enteric aHUS associated withan S. pneumonia infection may be treated with the compositions of thepresent invention.

In a still further aspect of the present invention, a subject sufferingfrom aHUS may initially be treated with a MASP-2 inhibitory compositionof the present invention that is administered through a catheter line,such as an intravenous catheter line or a subcutaneous catheter line,for a first period of time such as one hour, twelve hours, one day, twodays or three days. The subject may then be treated for a second periodof time with the MASP-2 inhibitory composition administered throughregular subcutaneous injections, such as daily, biweekly, weekly, everyother week, monthly or bimonthly, injections.

In a still further aspect of the present invention, a MASP-2 inhibitorycomposition of the present invention may be administered to a subjectsuffering from aHUS in the absence of plasmapheresis (i.e., a subjectwhose aHUS symptoms have not been treated with plasmapheresis and arenot treated with plasmapheresis at the time of treatment with the MASP-2inhibitory composition), to avoid the potential complications ofplasmaphersis including hemorrhage, infection, and exposure to disordersand/or allergies inherent in the plasma donor, or in a subject otherwiseaverse to plasmapheresis, or in a setting where plasmapheresis isunavailable.

In a still further aspect of the present invention, a MASP-2 inhibitorycomposition of the present invention may be administered to a subjectsuffering from aHUS coincident with treating the patient withplasmapheresis. For example, a subject receiving plasmapheresistreatment can then be administered the MASP-2 inhibitory compositionfollowing or alternating with plasma exchange.

In a still further aspect of the present invention, a subject sufferingfrom or at risk of developing aHUS and being treated with a MASP-2inhibitory composition of the present invention can be monitored byperiodically determining, such as every twelve hours or on a dailybasis, the level of at least one complement factor, wherein thedetermination of a reduced level of the at least one complement factorin comparison to a standard value or to a healthy subject is indicativeof the need for continued treatment with the composition.

In both prophylactic and therapeutic regimens, compositions comprisingMASP-2 inhibitory agents may be administered in several dosages until asufficient therapeutic outcome has been achieved in the subject. In oneembodiment of the invention, the MASP-2 inhibitory agent comprises ananti-MASP-2 antibody, which suitably may be administered to an adultpatient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still moresuitably from 50.0 mg to 500 mg. For pediatric patients, dosage can beadjusted in proportion to the patient's weight. Application of theMASP-2 inhibitory compositions of the present invention may be carriedout by a single administration of the composition, or a limited sequenceof administrations, for treatment of aHUS. Alternatively, thecomposition may be administered at periodic intervals, such as daily,biweekly, weekly, every other week, monthly or bimonthly, over anextended period of time for treatment of aHUS.

In some embodiments, the subject suffering from aHUS has previouslyundergone, or is currently undergoing treatment with a terminalcomplement inhibitor that inhibits cleavage of complement protein C5. Insome embodiments, the method comprises administering to the subject acomposition of the invention comprising a MASP-2 inhibitor and furtheradministering to the subject a terminal complement inhibitor thatinhibits cleavage of complement protein C5. In some embodiments, theterminal complement inhibitor is a humanized anti-C5 antibody orantigen-binding fragment thereof. In some embodiments, the terminalcomplement inhibitor is eculizumab.

In one aspect of the invention, the pharmaceutical compositions areadministered to a subject susceptible to, or otherwise at risk of, HUSin an amount sufficient to eliminate or reduce the risk of developingsymptoms of the condition. In therapeutic applications, thepharmaceutical compositions are administered to a subject suspected of,or already suffering from, HUS in a therapeutically effective amountsufficient to relieve, or at least partially reduce, the symptoms of thecondition.

In another aspect of the present invention, the likelihood of developingimpaired renal function in a subject at risk for developing HUS can bereduced by administering to the subject a MASP-2 inhibitory compositionof the present invention in an amount effective to inhibit MASP-2dependent complement activation. For example, a subject at risk fordeveloping HUS and to be treated with a MASP-2 inhibitory composition ofthe present invention may exhibit one or more symptoms associated withHUS, including diarrhea, a hematocrit level of less than 30% with smearevidence of intravascular erythrocyte destruction, thrombocytopenia andrising creatinine levels. As a further example, a subject at risk fordeveloping HUS and to be treated with the MASP-2 inhibitory compositionsof the present invention may be infected with E. coli, shigella orsalmonella. Such subjects infected with E. coli, shigella or salmonellamay be treated with a MASP-2 inhibitory composition of the presentinvention concurrent with antibiotic treatment, or alternately may betreated with a MASP-2 inhibitory composition without concurrenttreatment with an antibiotic, particularly for enterogenic E. coli forwhich antibiotic treatment is contra-indicated. A subject infected withenterogenic E. coli that has been treated with an antibiotic may be atelevated risk of developing HUS, and may be suitably treated with aMASP-2 inhibitory composition of the present invention to reduce thatrisk. A subject infected with enterogenic E. coli may be treated for afirst period of time with a MASP-2 inhibitory composition of the presentinvention in the absence of an antibiotic and then for a second periodof time with both a MASP-2 inhibitory composition of the presentinvention and an antibiotic.

In a still further aspect of the present invention, a subject sufferingfrom HUS may initially be treated with a MASP-2 inhibitory compositionof the present invention that is administered through a catheter line,such as an intravenous catheter line or a subcutaneous catheter line,for a first period of time such as one hour, twelve hours, one day, twodays or three days. The subject may then be treated for a second periodof time with the MASP-2 inhibitory composition administered throughregular subcutaneous injections, such as daily, biweekly, weekly, everyother week, monthly or bimonthly, injections.

In a still further aspect of the present invention, a MASP-2 inhibitorycomposition of the present invention may be administered to a subjectsuffering from HUS in the absence of plasmapheresis (i.e., a subjectwhose HUS symptoms have not been treated with plasmapheresis and are nottreated with plasmapheresis at the time of treatment with the MASP-2inhibitory composition), to avoid the potential complications ofplasmaphersis including hemorrhage, infection, and exposure to disordersand/or allergies inherent in the plasma donor, or in a subject otherwiseaverse to plasmapheresis, or in a setting where plasmapheresis isunavailable.

In a still further aspect of the present invention, a MASP-2 inhibitorycomposition of the present invention may be administered to a subjectsuffering from HUS coincident with treating the patient withplasmapheresis. For example, a subject receiving plasmapheresistreatment can then be administered the MASP-2 inhibitory compositionfollowing or alternating with plasma exchange.

In a still further aspect of the present invention, a subject sufferingfrom or at risk of developing HUS and being treated with a MASP-2inhibitory composition of the present invention can be monitored byperiodically determining, such as every twelve hours or on a dailybasis, the level of at least one complement factor, wherein thedetermination of a reduced level of the at least one complement factorin comparison to a standard value or to a healthy subject is indicativeof the need for continued treatment with the composition.

In both prophylactic and therapeutic regimens, compositions comprisingMASP-2 inhibitory agents may be administered in several dosages until asufficient therapeutic outcome has been achieved in the subject. In oneembodiment of the invention, the MASP-2 inhibitory agent comprises ananti-MASP-2 antibody, which suitably may be administered to an adultpatient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still moresuitably from 50.0 mg to 500 mg. For pediatric patients, dosage can beadjusted in proportion to the patient's weight. Application of theMASP-2 inhibitory compositions of the present invention may be carriedout by a single administration of the composition, or a limited sequenceof administrations, for treatment of HUS. Alternatively, the compositionmay be administered at periodic intervals, such as daily, biweekly,weekly, every other week, monthly or bimonthly, over an extended periodof time for treatment of HUS.

In some embodiments, the subject suffering from HUS has previouslyundergone, or is currently undergoing treatment with a terminalcomplement inhibitor that inhibits cleavage of complement protein C5. Insome embodiments, the method comprises administering to the subject acomposition of the invention comprising a MASP-2 inhibitor and furtheradministering to the subject a terminal complement inhibitor thatinhibits cleavage of complement protein C5. In some embodiments, theterminal complement inhibitor is a humanized anti-C5 antibody orantigen-binding fragment thereof. In some embodiments, the terminalcomplement inhibitor is eculizumab.

In one aspect of the invention, the pharmaceutical compositions areadministered to a subject susceptible to, or otherwise at risk of, TTPin an amount sufficient to eliminate or reduce the risk of developingsymptoms of the condition. In therapeutic applications, thepharmaceutical compositions are administered to a subject suspected of,or already suffering from, TTP in a therapeutically effective amountsufficient to relieve, or at least partially reduce, the symptoms of thecondition.

In another aspect of the present invention, a subject exhibiting one ormore of the symptoms of TTP, including central nervous systeminvolvement, thrombocytopenia, severe cardiac involvement, severepulmonary involvement, gastro-intestinal infarction and gangrene, may betreated with a MASP-2 inhibitory composition of the present invention.In another aspect of the present invention, a subject determined to havea depressed level of ADAMTS13 and also testing positive for the presenceof an inhibitor of (i.e., an antibody) ADAMTS 13 may be treated with aMASP-2 inhibitory composition of the present invention. In a stillfurther aspect of the present invention, a subject testing positive forthe presence of an inhibitor of ADAMTS13 may be treated with animmunosupressant (e.g., corticosteroids, rituxan, or cyclosporine)concurrently with treatment with a MASP-2 inhibitory composition of thepresent invention. In a still further aspect of the present invention, asubject determined to have a reduced level of ADAMTS13 and testingpositive for the presence of an inhibitor of ADAMTS13 may be treatedwith ADAMTS13 concurrently with treatment with a MASP-2 inhibitorycomposition of the present invention.

In a still further aspect of the present invention, a subject sufferingfrom TTP may initially be treated with a MASP-2 inhibitory compositionof the present invention that is administered through a catheter line,such as an intravenous catheter line or a subcutaneous catheter line,for a first period of time such as one hour, twelve hours, one day, twodays or three days. The subject may then be treated for a second periodof time with the MASP-2 inhibitory composition administered throughregular subcutaneous injections, such as daily, biweekly, weekly, everyother week, monthly or bimonthly, injections.

In a still further aspect of the present invention, a MASP-2 inhibitorycomposition of the present invention may be administered to a subjectsuffering from HUS in the absence of plasmapheresis (i.e., a subjectwhose TTP symptoms have not been treated with plasmapheresis and are nottreated with plasmapheresis at the time of treatment with the MASP-2inhibitory composition), to avoid the potential complications ofplasmaphersis including hemorrhage, infection, and exposure to disordersand/or allergies inherent in the plasma donor, or in a subject otherwiseaverse to plasmapheresis, or in a setting where plasmapheresis isunavailable.

In a still further aspect of the present invention, a MASP-2 inhibitorycomposition of the present invention may be administered to a subjectsuffering from TTP coincident with treating the patient withplasmapheresis. For example, a subject receiving plasmapheresistreatment can then be administered the MASP-2 inhibitory compositionfollowing or alternating with plasma exchange.

In a still further aspect of the present invention, a subject sufferingfrom refractory TTP, i.e., symptoms of TTP that have not respondedadequately to other treatment such as plasmapheresis, may be treatedwith a MASP-2 inhibitory composition of the present invention, with orwithout additional plasmapheresis.

In a still further aspect of the present invention, a subject sufferingfrom or at risk of developing TTP and being treated with a MASP-2inhibitory composition of the present invention can be monitored byperiodically determining, such as every twelve hours or on a dailybasis, the level of at least one complement factor, wherein thedetermination of a reduced level of the at least one complement factorin comparison to a standard value or to a healthy subject is indicativeof the need for continued treatment with the composition.

In both prophylactic and therapeutic regimens, compositions comprisingMASP-2 inhibitory agents may be administered in several dosages until asufficient therapeutic outcome has been achieved in the subject. In oneembodiment of the invention, the MASP-2 inhibitory agent comprises ananti-MASP-2 antibody, which suitably may be administered to an adultpatient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still moresuitably from 50.0 mg to 500 mg. For pediatric patients, dosage can beadjusted in proportion to the patient's weight. Application of theMASP-2 inhibitory compositions of the present invention may be carriedout by a single administration of the composition, or a limited sequenceof administrations, for treatment of TTP. Alternatively, the compositionmay be administered at periodic intervals, such as daily, biweekly,weekly, every other week, monthly or bimonthly, over an extended periodof time for treatment of TTP.

In some embodiments, the subject suffering from TTP has previouslyundergone, or is currently undergoing treatment with a terminalcomplement inhibitor that inhibits cleavage of complement protein C5. Insome embodiments, the method comprises administering to the subject acomposition of the invention comprising a MASP-2 inhibitor and furtheradministering to the subject a terminal complement inhibitor thatinhibits cleavage of complement protein C5. In some embodiments, theterminal complement inhibitor is a humanized anti-C5 antibody orantigen-binding fragment thereof. In some embodiments, the terminalcomplement inhibitor is eculizumab.

VI. EXAMPLES

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention. All literature citations herein are expressly incorporated byreference.

Example 1

This example describes the generation of a mouse strain deficient inMASP-2 (MASP-2−/−) but sufficient ofMAp19 (MAp19+/+).

Materials and Methods:

The targeting vector pKO-NTKV 1901 was designed to disrupt the threeexons coding for the C-terminal end of murine MASP-2, including the exonthat encodes the serine protease domain, as shown in FIG. 3. PKO-NTKV1901 was used to transfect the murine ES cell line E14.1a (SV129 Ola).Neomycin-resistant and Thymidine Kinase-sensitive clones were selected.600 ES clones were screened and, of these, four different clones wereidentified and verified by southern blot to contain the expectedselective targeting and recombination event as shown in FIG. 3. Chimeraswere generated from these four positive clones by embryo transfer. Thechimeras were then backcrossed in the genetic background C57/BL6 tocreate transgenic males. The transgenic males were crossed with femalesto generate F1s with 50% of the offspring showing heterozygosity for thedisrupted MASP-2 gene. The heterozygous mice were intercrossed togenerate homozygous MASP-2 deficient offspring, resulting inheterozygous and wild-type mice in the ration of 1:2:1, respectively.

Results and Phenotype:

The resulting homozygous MASP-2−/− deficient mice were found to beviable and fertile and were verified to be MASP-2 deficient by southernblot to confirm the correct targeting event, by Northern blot to confirmthe absence of MASP-2 mRNA, and by Western blot to confirm the absenceof MASP-2 protein (data not shown). The presence of MAp19 mRNA and theabsence of MASP-2 mRNA were further confirmed using time-resolved RT-PCRon a LightCycler machine. The MASP-2−/− mice do continue to expressMAp19, MASP-1, and MASP-3 mRNA and protein as expected (data not shown).The presence and abundance of mRNA in the MASP-2−/− mice for Properdin,Factor B, Factor D, C4, C2, and C3 was assessed by LightCycler analysisand found to be identical to that of the wild-type littermate controls(data not shown). The plasma from homozygous MASP-2−/− mice is totallydeficient of lectin-pathway-mediated complement activation as furtherdescribed in Example 2.

Generation of a MASP-2−/− strain on a pure C57BL6 Background: TheMASP-2−/− mice were back-crossed with a pure C57BL6 line for ninegenerations prior to use of the MASP-2−/− strain as an experimentalanimal model.

A transgenic mouse strain that is murine MASP-2−/−, MAp19+/+ and thatexpresses a human MASP-2 transgene (a murine MASP-2 knock-out and ahuman MASP-2 knock-in) was also generated as follows:

Materials and Methods:

A minigene encoding human MASP-2 called “mini hMASP-2” (SEQ ID NO:49) asshown in FIG. 4 was constructed which includes the promoter region ofthe human MASP 2 gene, including the first 3 exons (exon 1 to exon 3)followed by the cDNA sequence that represents the coding sequence of thefollowing 8 exons, thereby encoding the full-length MASP-2 proteindriven by its endogenous promoter. The mini hMASP-2 construct wasinjected into fertilized eggs of MASP-2−/− in order to replace thedeficient murine MASP 2 gene by transgenically expressed human MASP-2.

Example 2

This example demonstrates that MASP-2 is required for complementactivation via the lectin pathway.

Methods and Materials:

Lectin Pathway Specific C4 Cleavage Assay:

A C4 cleavage assay has been described by Petersen, et al., J. Immunol.Methods 257:107 (2001) that measures lectin pathway activation resultingfrom lipoteichoic acid (LTA) from S. aureus, which binds L-ficolin. Theassay described by Petersen et al., (2001) was adapted to measure lectinpathway activation via MBL by coating the plate with LPS and mannan orzymosan prior to adding serum from MASP-2 −/− mice as described below.The assay was also modified to remove the possibility of C4 cleavage dueto the classical pathway. This was achieved by using a sample dilutionbuffer containing 1 M NaCl, which permits high affinity binding oflectin pathway recognition components to their ligands but preventsactivation of endogenous C4, thereby excluding the participation of theclassical pathway by dissociating the C1 complex. Briefly described, inthe modified assay serum samples (diluted in high salt (1 M NaCl)buffer) are added to ligand-coated plates, followed by the addition of aconstant amount of purified C4 in a buffer with a physiologicalconcentration of salt. Bound recognition complexes containing MASP-2cleave the C4, resulting in C4b deposition.

Assay Methods:

1) Nunc Maxisorb microtiter plates (Maxisorb, Nunc, Cat. No. 442404,Fisher Scientific) were coated with 1 μg/ml mannan (M7504 Sigma) or anyother ligand (e.g., such as those listed below) diluted in coatingbuffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6).

The following reagents were used in the assay:

-   -   a. mannan (1 μg/well mannan (M7504 Sigma) in 100 jpl coating        buffer):    -   b. zymosan (1 μg/well zymosan (Sigma) in 100 μl coating buffer);    -   c. LTA (1 g/well in 100 μl coating buffer or 2 μg/well in 20 μl        methanol)    -   d. 1 μg of the H-ficolin specific Mab 4H5 in coating buffer    -   e. PSA from Aerococcus viridans (2 μg/well in 100 μl coating        buffer)    -   f. 100 μl/well of formalin-fixed S. aureus DSM20233 (OD₅₅₀=0.5)        in coating buffer.

2) The plates were incubated overnight at 4° C.

3) After overnight incubation, the residual protein binding sites weresaturated by incubated the plates with 0.1% HSA-TBS blocking buffer(0.1% (w/v) HSA in 10 mM Tris-CL, 140 mM NaCl, 1.5 mM NaN₃, pH 7.4) for1-3 hours, then washing the plates 3× with TBS/tween/Ca²⁺ (TBS with0.05% Tween 20 and 5 mM CaCl₂, 1 mM MgCl₂, pH 7.4).

4) Serum samples to be tested were diluted in MBL-binding buffer (1 MNaCl) and the diluted samples were added to the plates and incubatedovernight at 4° C. Wells receiving buffer only were used as negativecontrols.

5) Following incubation overnight at 4° C., the plates were washed 3×with TBS/tween/Ca2+. Human C4 (100 μl/well of 1 μg/ml diluted in BBS (4mM barbital, 145 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4)) was thenadded to the plates and incubated for 90 minutes at 37° C. The plateswere washed again 3× with TBS/tween/Ca²⁺.

6) C4b deposition was detected with an alkaline phosphatase-conjugatedchicken anti-human C4c (diluted 1:1000 in TBS/tween/Ca²⁺), which wasadded to the plates and incubated for 90 minutes at room temperature.The plates were then washed again 3× with TBS/tween/Ca²⁺.

7) Alkaline phosphatase was detected by adding 100 μl of p-nitrophenylphosphate substrate solution, incubating at room temperature for 20minutes, and reading the OD₄₀₅ in a microtiter plate reader.

Results:

FIGS. 5A-B show the amount of C4b deposition on mannan (FIG. 5A) andzymosan (FIG. 5B) in serum dilutions from MASP-2+/+(crosses),MASP-2+/−(closed circles) and MASP-2−/− (closed triangles). FIG. 5Cshows the relative C4 convertase activity on plates coated with zymosan(white bars) or mannan (shaded bars) from MASP-2−/+mice (n=5) andMASP-2−/− mice (n=4) relative to wild-type mice (n=5) based on measuringthe amount of C4b deposition normalized to wild-type serum. The errorbars represent the standard deviation. As shown in FIGS. 5A-C, plasmafrom MASP-2−/− mice is totally deficient in lectin-pathway-mediatedcomplement activation on mannan and on zymosan coated plates. Theseresults clearly demonstrate that MASP-2 is an effector component of thelectin pathway.

Recombinant MASP-2 Reconstitutes Lectin Pathway-Dependent C4 Activationin Serum from the MASP-2−/− Mice

In order to establish that the absence of MASP-2 was the direct cause ofthe loss of lectin pathway-dependent C4 activation in the MASP-2−/−mice, the effect of adding recombinant MASP-2 protein to serum sampleswas examined in the C4 cleavage assay described above. Functionallyactive murine MASP-2 and catalytically inactive murine MASP-2A (in whichthe active-site serine residue in the serine protease domain wassubstituted for the alanine residue) recombinant proteins were producedand purified as described below in Example 3. Pooled serum from 4 MASP-2−/− mice was pre-incubated with increasing protein concentrations ofrecombinant murine MASP-2 or inactive recombinant murine MASP-2A and C4convertase activity was assayed as described above.

Results:

As shown in FIG. 6, the addition of functionally active murinerecombinant MASP-2 protein (shown as open triangles) to serum obtainedfrom the MASP-2 −/− mice restored lectin pathway-dependent C4 activationin a protein concentration dependent manner, whereas the catalyticallyinactive murine MASP-2A protein (shown as stars) did not restore C4activation. The results shown in FIG. 6 are normalized to the C4activation observed with pooled wild-type mouse serum (shown as a dottedline).

Example 3

This example describes the recombinant expression and protein productionof recombinant full-length human, rat and murine MASP-2, MASP-2 derivedpolypeptides, and catalytically inactivated mutant forms of MASP-2

Expression of Full-Length Human, Murine and Rat MASP-2:

The full length cDNA sequence of human MASP-2 (SEQ ID NO: 4) was alsosubcloned into the mammalian expression vector pCI-Neo (Promega), whichdrives eukaryotic expression under the control of the CMVenhancer/promoter region (described in Kaufman R. J. et al., NucleicAcids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology,185:537-66 (1991)). The full length mouse cDNA (SEQ ID NO:50) and ratMASP-2 cDNA (SEQ ID NO:53) were each subcloned into the pED expressionvector. The MASP-2 expression vectors were then transfected into theadherent Chinese hamster ovary cell line DXB1 using the standard calciumphosphate transfection procedure described in Maniatis et al., 1989.Cells transfected with these constructs grew very slowly, implying thatthe encoded protease is cytotoxic.

In another approach, the minigene construct (SEQ ID NO:49) containingthe human cDNA of MASP-2 driven by its endogenous promoter istransiently transfected into Chinese hamster ovary cells (CHO). Thehuman MASP-2 protein is secreted into the culture media and isolated asdescribed below.

Expression of Full-Length Catalytically Inactive MASP-2:

Rationale: MASP-2 is activated by autocatalytic cleavage after therecognition subcomponents MBL or ficolins (either L-ficolin, H-ficolinor M-ficolin) bind to their respective carbohydrate pattern.Autocatalytic cleavage resulting in activation of MASP-2 often occursduring the isolation procedure of MASP-2 from serum, or during thepurification following recombinant expression. In order to obtain a morestable protein preparation for use as an antigen, a catalyticallyinactive form of MASP-2, designed as MASP-2A was created by replacingthe serine residue that is present in the catalytic triad of theprotease domain with an alanine residue in rat (SEQ ID NO:55 Ser617 toAla617); in mouse (SEQ ID NO:52 Ser617 to Ala617); or in human (SEQ IDNO:3 Ser618 to Ala618).

In order to generate catalytically inactive human and murine MASP-2Aproteins, site-directed mutagenesis was carried out using theoligonucleotides shown in TABLE 5. The oligonucleotides in TABLE 5 weredesigned to anneal to the region of the human and murine cDNA encodingthe enzymatically active serine and oligonucleotide contain a mismatchin order to change the serine codon into an alanine codon. For example,PCR oligonucleotides SEQ ID NOS:56-59 were used in combination withhuman MASP-2 cDNA (SEQ ID NO:4) to amplify the region from the startcodon to the enzymatically active serine and from the serine to the stopcodon to generate the complete open reading from of the mutated MASP-2Acontaining the Ser618 to Ala618 mutation. The PCR products were purifiedafter agarose gel electrophoresis and band preparation and singleadenosine overlaps were generated using a standard tailing procedure.The adenosine tailed MASP-2A was then cloned into the pGEM-T easyvector, transformed into E. coli.

A catalytically inactive rat MASP-2A protein was generated by kinasingand annealing SEQ ID NO:64 and SEQ ID NO:65 by combining these twooligonucleotides in equal molar amounts, heating at 100° C. for 2minutes and slowly cooling to room temperature. The resulting annealedfragment has Pstl and Xbal compatible ends and was inserted in place ofthe Pstl-Xbal fragment of the wild-type rat MASP-2 cDNA (SEQ ID NO:53)to generate rat MASP-2A.

(SEQ ID NO: 64) 5 ′GAGGTGACGCAGGAGGGGCATTAGTGTTT 3′ (SEQ ID NO: 65)5′ CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3′

The human, murine and rat MASP-2A were each further subcloned intoeither of the mammalian expression vectors pED or pCI-Neo andtransfected into the Chinese Hamster ovary cell line DXB 1 as describedbelow.

In another approach, a catalytically inactive form of MASP-2 isconstructed using the method described in Chen et al., J. Biol. Chem.,276(28):25894-25902, 2001. Briefly, the plasmid containing thefull-length human MASP-2 cDNA (described in Thiel et al., Nature386:506, 1997) is digested with Xho1 and EcoR1 and the MASP-2 cDNA(described herein as SEQ ID NO:4) is cloned into the correspondingrestriction sites of the pFastBacl baculovirus transfer vector (LifeTechnologies, NY). The MASP-2 serine protease active site at Ser618 isthen altered to Ala618 by substituting the double-strandedoligonucleotides encoding the peptide region amino acid 610-625 (SEQ IDNO:13) with the native region amino acids 610 to 625 to create a MASP-2full length polypeptide with an inactive protease domain. Constructionof Expression Plasmids Containing Polypeptide Regions Derived from HumanMasp-2.

The following constructs are produced using the MASP-2 signal peptide(residues 1-15 of SEQ ID NO:5) to secrete various domains of MASP-2. Aconstruct expressing the human MASP-2 CUBI domain (SEQ ID NO:8) is madeby PCR amplifying the region encoding residues 1-121 of MASP-2 (SEQ IDNO:6) (corresponding to the N-terminal CUB 1 domain). A constructexpressing the human MASP-2 CUBIEGF domain (SEQ ID NO:9) is made by PCRamplifying the region encoding residues 1-166 of MASP-2 (SEQ ID NO:6)(corresponding to the N-terminal CUBIEGF domain). A construct expressingthe human MASP-2 CUBIEGFCUBII domain (SEQ ID NO: 10) is made by PCRamplifying the region encoding residues 1-293 of MASP-2 (SEQ ID NO:6)(corresponding to the N-terminal CUBIEGFCUBII domain). The abovementioned domains are amplified by PCR using Vent_(R) polymerase andpBS-MASP-2 as a template, according to established PCR methods. The 5′primer sequence of the sense primer (5′-CGGGATCCATGAGGCTGCTGACCCTC-3′SEQ ID NO:34) introduces a BamHI restriction site (underlined) at the 5′end of the PCR products. Antisense primers for each of the MASP-2domains, shown below in TABLE 5, are designed to introduce a stop codon(boldface) followed by an EcoRI site (underlined) at the end of each PCRproduct. Once amplified, the DNA fragments are digested with BamHI andEcoRI and cloned into the corresponding sites of the pFastBacl vector.The resulting constructs are characterized by restriction mapping andconfirmed by dsDNA sequencing.

TABLE 5 MASP-2 PCR PRIMERS MASP-2 domain 5′ PCR Primer 3′ PCR PrimerSEQ ID NO: 8 5′ CGGGATCC 5′ GGAATTCC CUBI ATGAGGCTGCT TAGGCTGCATA(aa 1-121 of GACCCTC-3′ (SEQ ID SEQ ID NO: 6) (SEQ ID NO: 35) NO: 34)SEQ ID NO: 9 5′ CGGGATCC 5′ GGAATTCC CUBIEGF ATGAGGCTGCT TACAGGGCG(aa 1-166 of GACCCTC-3′ T-3′ SEQ ID NO: 6) (SEQ ID (SEQ ID NO: 34)NO: 36) SEQ ID NO: 10 5′ CGGGATCC 5′ GGAATTCCT CUBIEGFCUBII ATGAGGCTGCTAGTAGT (aa 1-293 of GACCCTC-3′ GGAT 3′ SEQ ID NO: 6) (SEQ ID (SEQ IDNO: 34) NO: 37) SEQ ID NO: 4 5′ ATGAGGCT 5′ TTAAAATCA human MASP-2GCTGACCCTCC CTAATTATGTTC TGGGCCTTC TCGATC 3′ 3′(SEQ ID (SEQ ID NO: 56)NO: 59) hMASP-2_ hMASP-2_ forward reverse SEQ ID NO: 4 5′ CAGAGGTG5′ GTGCCCCTC human MASP-2 ACGCAGGAGGG CTGCGTCACCTC cDNA GCAC 3′ TG 3′(SEQ ID (SEQ ID NO: 58) NO: 57) hMASP-2_ala_ hMASP-2_ala_ forwardreverse SEQ ID NO: 50 5′ ATGAGGCT 5′ TTAGAAATT Murine MASP-2 ACTCATCTTCCACTTATTATGT cDNA TGG 3′ TCTCAATCC 3′ (SEQ ID (SEQ ID NO: 60) NO: 63)mMASP-2_ mMASP-2_ forward reverse SEQ ID NO: 50 5′ CCCCCCCT 5′ CTGCAGAGMurine MASP-2 GCGTCACCTCT GTGACGCAGGG cDNA GCAG 3′ GGGG 3′ (SEQ ID(SEQ ID NO: 62) NO: 61) mMASP-2_ala_ mMASP-2_ala_ forward reverse

Recombinant Eukaryotic Expression of MASP-2 and Protein Production ofEnzymatically Inactive Mouse, Rat, and Human MASP-2A.

The MASP-2 and MASP-2A expression constructs described above weretransfected into DXB 1 cells using the standard calcium phosphatetransfection procedure (Maniatis et al., 1989). MASP-2A was produced inserum-free medium to ensure that preparations were not contaminated withother serum proteins. Media was harvested from confluent cells everysecond day (four times in total). The level of recombinant MASP-2Aaveraged approximately 1.5 mg/liter of culture medium for each of thethree species.

MASP-2A Protein Purification:

The MASP-2A (Ser-Ala mutant described above) was purified by affinitychromatography on MBP-A-agarose columns. This strategy enabled rapidpurification without the use of extraneous tags. MASP-2A (100-200 ml ofmedium diluted with an equal volume of loading buffer (50 mM Tris-Cl, pH7.5, containing 150 mM NaCl and 25 mM CaCl₂) was loaded onto anMBP-agarose affinity column (4 ml) pre-equilibrated with 10 ml ofloading buffer. Following washing with a further 10 ml of loadingbuffer, protein was eluted in 1 ml fractions with 50 mM Tris-Cl, pH 7.5,containing 1.25 M NaCl and 10 mM EDTA. Fractions containing the MASP-2Awere identified by SDS-polyacrylamide gel electrophoresis. Wherenecessary, MASP-2A was purified further by ion-exchange chromatographyon a MonoQ column (HR 5/5). Protein was dialysed with 50 mM Tris-Cl pH7.5, containing 50 mM NaCl and loaded onto the column equilibrated inthe same buffer. Following washing, bound MASP-2A was eluted with a0.05-1 M NaCl gradient over 10 ml.

Results:

Yields of 0.25-0.5 mg of MASP-2A protein were obtained from 200 ml ofmedium. The molecular mass of 77.5 kDa determined by MALDI-MS is greaterthan the calculated value of the unmodified polypeptide (73.5 kDa) dueto glycosylation. Attachment of glycans at each of the N-glycosylationsites accounts for the observed mass. MASP-2A migrates as a single bandon SDS-polyacrylamide gels, demonstrating that it is not proteolyticallyprocessed during biosynthesis. The weight-average molecular massdetermined by equilibrium ultracentrifugation is in agreement with thecalculated value for homodimers of the glycosylated polypeptide.

Production of Recombinant Human MASP-2 Polypeptides

Another method for producing recombinant MASP-2 and MASP2A derivedpolypeptides is described in Thielens, N. M., et al., J. Immunol.166:5068-5077, 2001. Briefly, the Spodoptera frugiperda insect cells(Ready-Plaque Sf9 cells obtained from Novagen, Madison, Wis.) are grownand maintained in Sf900II serum-free medium (Life Technologies)supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin (LifeTechnologies). The Trichoplusia ni (High Five) insect cells (provided byJadwiga Chroboczek, Institut de Biologie Structurale, Grenoble, France)are maintained in TC100 medium (Life Technologies) containing 10% FCS(Dominique Dutscher, Brumath, France) supplemented with 50 IU/mlpenicillin and 50 mg/ml streptomycin. Recombinant baculoviruses aregenerated using the Bac-to-Bac system (Life Technologies). The bacmidDNA is purified using the Qiagen midiprep purification system (Qiagen)and is used to transfect Sf9 insect cells using cellfectin in Sf900 IISFM medium (Life Technologies) as described in the manufacturer'sprotocol. Recombinant virus particles are collected 4 days later,titrated by virus plaque assay, and amplified as described by King andPossee, in The Baculovirus Expression System: A Laboratory Guide,Chapman and Hall Ltd., London, pp. 111-114, 1992.

High Five cells (1.75×10⁷ cells/175-cm² tissue culture flask) areinfected with the recombinant viruses containing MASP-2 polypeptides ata multiplicity of infection of 2 in SfO0 II SFM medium at 28° C. for 96h. The supernatants are collected by centrifugation and diisopropylphosphorofluoridate is added to a final concentration of 1 mM.

The MASP-2 polypeptides are secreted in the culture medium. The culturesupernatants are dialyzed against 50 mM NaCl, 1 mM CaCI₂, 50 mMtriethanolamine hydrochloride, pH 8.1, and loaded at 1.5 ml/min onto aQ-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8×12 cm)equilibrated in the same buffer. Elution is conducted by applying al.2liter linear gradient to 350 mM NaCl in the same buffer. Fractionscontaining the recombinant MASP-2 polypeptides are identified by Westernblot analysis, precipitated by addition of (NH₄)₂SO₄ to 60% (w/v), andleft overnight at 4° C. The pellets are resuspended in 145 mM NaCl, 1 mMCaCl₂, 50 mM triethanolamine hydrochloride, pH 7.4, and applied onto aTSK G3000 SWG column (7.5×600 mm) (Tosohaas, Montgomeryville, Pa.)equilibrated in the same buffer. The purified polypeptides are thenconcentrated to 0.3 mg/ml by ultrafiltration on Microsepmicroconcentrators (m.w. cut-off=10,000) (Filtron, Karlstein, Germany).

Example 4

This example describes a method of producing polyclonal antibodiesagainst MASP-2 polypeptides.

Materials and Methods:

MASP-2 Antigens:

Polyclonal anti-human MASP-2 antiserum is produced by immunizing rabbitswith the following isolated MASP-2 polypeptides: human MASP-2 (SEQ IDNO:6) isolated from serum; recombinant human MASP-2 (SEQ ID NO:6),MASP-2A containing the inactive protease domain (SEQ ID NO:13), asdescribed in Example 3; and recombinant CUBI (SEQ ID NO:8), CUBEGFI (SEQID NO:9), and CUBEGFCUBII (SEQ ID NO: 10) expressed as described abovein Example 3.

Polyclonal Antibodies:

Six-week old Rabbits, primed with BCG (bacillus Calmette-Guerin vaccine)are immunized by injecting 100 gig of MASP-2 polypeptide at 100 μg/ml insterile saline solution. Injections are done every 4 weeks, withantibody titer monitored by ELISA assay as described in Example 5.Culture supernatants are collected for antibody purification by proteinA affinity chromatography.

Example 5

This example describes a method for producing murine monoclonalantibodies against rat or human MASP-2 polypeptides.

Materials and Methods:

Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks old, are injectedsubcutaneously with 100 gig human or rat rMASP-2 or rMASP-2Apolypeptides (made as described in Example 3) in complete Freund'sadjuvant (Difco Laboratories, Detroit, Mich.) in 200 pj of phosphatebuffered saline (PBS) pH 7.4. At two-week intervals the mice are twiceinjected subcutaneously with 50 jg of human or rat rMASP-2 or rMASP-2Apolypeptide in incomplete Freund's adjuvant. On the fourth week the miceare injected with 50 μg of human or rat rMASP-2 or rMASP-2A polypeptidein PBS and are fused 4 days later.

For each fusion, single cell suspensions are prepared from the spleen ofan immunized mouse and used for fusion with Sp2/0 myeloma cells. 5×10⁸of the Sp2/0 and 5×10⁸ spleen cells are fused in a medium containing 50%polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5%dimethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells arethen adjusted to a concentration of 1.5×10⁵ spleen cells per 200 μl ofthe suspension in Iscove medium (Gibco, Grand Island, N.Y.),supplemented with 10% fetal bovine serum, 100 units/ml of penicillin,100 μg/ml of streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin and16 μM thymidine. Two hundred microliters of the cell suspension areadded to each well of about twenty 96-well microculture plates. Afterabout ten days culture supernatants are withdrawn for screening forreactivity with purified factor MASP-2 in an ELISA assay.

ELISA Assay:

Wells of Immulon 2 (Dynatech Laboratories, Chantilly, Va.) microtestplates are coated by adding 50 μl of purified hMASP-2 at 50 ng/ml or ratrMASP-2 (or rMASP-2A) overnight at room temperature. The lowconcentration of MASP-2 for coating enables the selection ofhigh-affinity antibodies. After the coating solution is removed byflicking the plate, 200 μl of BLOTTO (non-fat dry milk) in PBS is addedto each well for one hour to block the non-specific sites. An hourlater, the wells are then washed with a buffer PBST (PBS containing0.05% Tween 20). Fifty microliters of culture supernatants from eachfusion well is collected and mixed with 50 μl of BLOTTO and then addedto the individual wells of the microtest plates. After one hour ofincubation, the wells are washed with PBST. The bound murine antibodiesare then detected by reaction with horseradish peroxidase (HRP)conjugated goat anti-mouse IgG (Fc specific) (Jackson ImmunoResearchLaboratories, West Grove, Pa.) and diluted at 1:2,000 in BLOTTO.Peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethylbenzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma)is added to the wells for color development for 30 minutes. The reactionis terminated by addition of 50 μl of 2M H₂SO₄ per well. The OpticalDensity at 450 nm of the reaction mixture is read with a BioTek ELISAReader (BioTek Instruments, Winooski, Vt.).

MASP-2 Binding Assay:

Culture supernatants that test positive in the MASP-2 ELISA assaydescribed above can be tested in a binding assay to determine thebinding affinity the MASP-2 inhibitory agents have for MASP-2. A similarassay can also be used to determine if the inhibitory agents bind toother antigens in the complement system.

Polystyrene microtiter plate wells (96-well medium binding plates,Corning Costar, Cambridge, Mass.) are coated with MASP-2 (20 ng/100μl/well, Advanced Research Technology, San Diego, Calif.) inphosphate-buffered saline (PBS) pH 7.4 overnight at 4° C. Afteraspirating the MASP-2 solution, wells are blocked with PBS containing 1%bovine serum albumin (BSA; Sigma Chemical) for 2 h at room temperature.Wells without MASP-2 coating serve as the background controls. Aliquotsof hybridoma supernatants or purified anti-MASP-2 MoAbs, at varyingconcentrations in blocking solution, are added to the wells. Following a2 h incubation at room temperature, the wells are extensively rinsedwith PBS. MASP-2-bound anti-MASP-2 MoAb is detected by the addition ofperoxidase-conjugated goat anti-mouse IgG (Sigma Chemical) in blockingsolution, which is allowed to incubate for 1h at room temperature. Theplate is rinsed again thoroughly with PBS, and 100 μl of3,3′,5,5′-tetramethyl benzidine (TMB) substrate (Kirkegaard and PerryLaboratories, Gaithersburg, Md.) is added. The reaction of TMB isquenched by the addition of 100 μl of 1M phosphoric acid, and the plateis read at 450 nm in a microplate reader (SPECTRA MAX 250, MolecularDevices, Sunnyvale, Calif.).

The culture supernatants from the positive wells are then tested for theability to inhibit complement activation in a functional assay such asthe C4 cleavage assay as described in Example 2. The cells in positivewells are then cloned by limiting dilution. The MoAbs are tested againfor reactivity with hMASP-2 in an ELISA assay as described above. Theselected hybridomas are grown in spinner flasks and the spent culturesupernatant collected for antibody purification by protein A affinitychromatography.

Example 6

This example describes the generation and production of humanized murineanti-MASP-2 antibodies and antibody fragments.

A murine anti-MASP-2 monoclonal antibody is generated in Male A/J miceas described in Example 5. The murine antibody is then humanized asdescribed below to reduce its immunogenicity by replacing the murineconstant regions with their human counterparts to generate a chimericIgG and Fab fragment of the antibody, which is useful for inhibiting theadverse effects of MASP-2-dependent complement activation in humansubjects in accordance with the present invention.

1. Cloning of Anti-MASP-2 Variable Region Genes from Murine HybridomaCells.

Total RNA is isolated from the hybridoma cells secreting anti-MASP-2MoAb (obtained as described in Example 7) using RNAzol following themanufacturer's protocol (Biotech, Houston, Tex.). First strand cDNA issynthesized from the total RNA using oligo dT as the primer. PCR isperformed using the immunoglobulin constant C region-derived 3′ primersand degenerate primer sets derived from the leader peptide or the firstframework region of murine V_(H) or V_(K) genes as the 5′ primers.Anchored PCR is carried out as described by Chen and Platsucas (Chen, P.F., Scand. J. Immunol. 35:539-549, 1992). For cloning the V_(K) gene,double-stranded cDNA is prepared using a Notl-MAK1 primer(5′-TGCGGCCGCTGTAGGTGCTGTCTTT-3′ SEQ ID NO:38). Annealed adaptors ADI(5′-GGAATTCACTCGTTATTCTCGGA-3′ SEQ ID NO:39) and AD2(5′-TCCGAGAATAACGAGTG-3′ SEQ ID NO:40) are ligated to both 5′ and 3′termini of the double-stranded cDNA. Adaptors at the 3′ ends are removedby Nod digestion. The digested product is then used as the template inPCR with the ADI oligonucleotide as the 5′ primer and MAK2(5′-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3′ SEQ ID NO:41) as the 3′ primer. DNAfragments of approximately 500 bp are cloned into pUC19. Several clonesare selected for sequence analysis to verify that the cloned sequenceencompasses the expected murine immunoglobulin constant region. TheNotl-MAK1 and MAK2 oligonucleotides are derived from the V_(K) regionand are 182 and 84 bp, respectively, downstream from the first base pairof the C kappa gene. Clones are chosen that include the complete V_(K)and leader peptide.

For cloning the V_(H) gene, double-stranded cDNA is prepared using theNotl MAGI primer (5′-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3′ SEQ ID NO:42).Annealed adaptors ADI and AD2 are ligated to both 5′ and 3′ termini ofthe double-stranded cDNA. Adaptors at the 3′ ends are removed by Noddigestion. The digested product are used as the template in PCR with theAD1 oligonucleotide and MAG2 (5′-CGGTAAGCTTCACTGGCTCAGGGAAATA-3′ SEQ IDNO:43) as primers. DNA fragments of 500 to 600 bp in length are clonedinto pUC19. The Notl-MAGI and MAG2 oligonucleotides are derived from themurine Cy.7.1 region, and are 180 and 93 bp, respectively, downstreamfrom the first bp of the murine Cy.7.1 gene. Clones are chosen thatencompass the complete V_(H) and leader peptide.

2. Construction of Expression Vectors for Chimeric MASP-2 IgG and Fab.

The cloned V_(H) and V_(K) genes described above are used as templatesin a PCR reaction to add the Kozak consensus sequence to the 5′ end andthe splice donor to the 3′ end of the nucleotide sequence. After thesequences are analyzed to confirm the absence of PCR errors, the V_(H)and V_(K) genes are inserted into expression vector cassettes containinghuman C.γ1 and C. kappa respectively, to give pSV2neoV_(H)-huCγ1 andpSV2neoV-huCγ. CsCl gradient-purified plasmid DNAs of the heavy- andlight-chain vectors are used to transfect COS cells by electroporation.After 48 hours, the culture supernatant is tested by ELISA to confirmthe presence of approximately 200 ng/ml of chimeric IgG. The cells areharvested and total RNA is prepared. First strand cDNA is synthesizedfrom the total RNA using oligo dT as the primer. This cDNA is used asthe template in PCR to generate the Fd and kappa DNA fragments. For theFd gene, PCR is carried out using5′-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3′ (SEQ ID NO:44) as the 5′primer and a CHI-derived 3′ primer(5′-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3′ SEQ ID NO:45). The DNA sequenceis confirmed to contain the complete V_(H) and the CH1 domain of humanIgG. After digestion with the proper enzymes, the Fd DNA fragments areinserted at the HindIII and BamHI restriction sites of the expressionvector cassette pSV2dhfr-TUS to give pSV2dhfrFd. The pSV2 plasmid iscommercially available and consists of DNA segments from varioussources: pBR322 DNA (thin line) contains the pBR322 origin of DNAreplication (pBR ori) and the lactamase ampicillin resistance gene(Amp); SV40 DNA, represented by wider hatching and marked, contains theSV40 origin of DNA replication (SV40 ori), early promoter (5′ to thedhfr and neo genes), and polyadenylation signal (3′ to the dhfr and neogenes). The SV40-derived polyadenylation signal (pA) is also placed atthe 3′ end of the Fd gene.

For the kappa gene, PCR is carried out using5′-AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3′ (SEQ ID NO:46) as the 5′ 5primer and a C_(K)-derived 3′ primer (5′-CGGGATCCTTCTCCCTCTAACACTCT-3′SEQ ID NO:47). DNA sequence is confirmed to contain the complete V_(K)and human C_(K) regions. After digestion with proper restrictionenzymes, the kappa DNA fragments are inserted at the HindIII and BamHIrestriction sites of the expression vector cassette pSV2neo-TUS to givepSV2neoK. The expression of both Fd and .kappa genes are driven by theHCMV-derived enhancer and promoter elements. Since the Fd gene does notinclude the cysteine amino acid residue involved in the inter-chaindisulfide bond, this recombinant chimeric Fab contains non-covalentlylinked heavy- and light-chains. This chimeric Fab is designated as cFab.

To obtain recombinant Fab with an inter-heavy and light chain disulfidebond, the above Fd gene may be extended to include the coding sequencefor additional 9 amino acids (EPKSCDKTH SEQ ID NO:48) from the hingeregion of human IgG. The BstEII-BamHI DNA segment encoding 30 aminoacids at the 3′ end of the Fd gene may be replaced with DNA segmentsencoding the extended Fd, resulting in pSV2dhfrFd/9aa.

3. Expression and Purification of Chimeric Anti-MASP-2 IgG

To generate cell lines secreting chimeric anti-MASP-2 IgG, NSO cells aretransfected with purified plasmid DNAs of pSV2neoV_(H)-huC.γ1 andpSV2neoV-huC kappa by electroporation. Transfected cells are selected inthe presence of 0.7 mg/ml G418. Cells are grown in a 250 ml spinnerflask using serum-containing medium.

Culture supernatant of 100 ml spinner culture is loaded on a 10-mlPROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column iswashed with 10 bed volumes of PBS. The bound antibody is eluted with 50mM citrate buffer, pH 3.0. Equal volume of 1 M Hepes, pH 8.0 is added tothe fraction containing the purified antibody to adjust the pH to 7.0.Residual salts are removed by buffer exchange with PBS by Milliporemembrane ultrafiltration (M.W. cut-off: 3,000). The proteinconcentration of the purified antibody is determined by the BCA method(Pierce).

4. Expression and Purification of Chimeric Anti-MASP-2 Fab

To generate cell lines secreting chimeric anti-MASP-2 Fab, CHO cells aretransfected with purified plasmid DNAs of pSV2dhfrFd (or pSV2dhfrFd/9aa)and pSV2neokappa, by electroporation. Transfected cells are selected inthe presence of G418 and methotrexate. Selected cell lines are amplifiedin increasing concentrations of methotrexate. Cells are single-cellsubcloned by limiting dilution. High-producing single-cell subclonedcell lines are then grown in 100 ml spinner culture using serum-freemedium.

Chimeric anti-MASP-2 Fab is purified by affinity chromatography using amouse anti-idiotypic MoAb to the MASP-2 MoAb. An anti-idiotypic MASP-2MoAb can be made by immunizing mice with a murine anti-MASP-2 MoAbconjugated with keyhole limpet hemocyanin (KLH) and screening forspecific MoAb binding that can be competed with human MASP-2. Forpurification, 100 ml of supernatant from spinner cultures of CHO cellsproducing cFab or cFab/9aa are loaded onto the affinity column coupledwith an anti-idiotype MASP-2 MoAb. The column is then washed thoroughlywith PBS before the bound Fab is eluted with 50 mM diethylamine, pH11.5. Residual salts are removed by buffer exchange as described above.The protein concentration of the purified Fab is determined by the BCAmethod (Pierce).

The ability of the chimeric MASP-2 IgG, cFab, and cFAb/9aa to inhibitMASP-2-dependent complement pathways may be determined by using theinhibitory assays described in Example 2 or Example 7.

Example 7

This example describes an in vitro C4 cleavage assay used as afunctional screen to identify MASP-2 inhibitory agents capable ofblocking MASP-2-dependent complement activation via L-ficolin/P35,H-ficolin, M-ficolin or mannan.

C4 Cleavage Assay:

A C4 cleavage assay has been described by Petersen, S. V., et al., J.Immunol. Methods 257:107, 2001, which measures lectin pathway activationresulting from lipoteichoic acid (LTA) from S. aureus which bindsL-ficolin.

Reagents:

Formalin-fixed S. aureous (DSM20233) is prepared as follows: bacteria isgrown overnight at 37° C. in tryptic soy blood medium, washed threetimes with PBS, then fixed for 1 h at room temperature in PBS/0.5%formalin, and washed a further three times with PBS, before beingresuspended in coating buffer (15 mM Na₂Co₃, 35 mM NaHCO₃, pH 9.6).

Assay:

The wells of a Nunc MaxiSorb microtiter plate (Nalgene NuncInternational, Rochester, N.Y.) are coated with: 100 μl offormalin-fixed S. aureus DSM20233 (OD550=0.5) in coating buffer with 1ug of L-ficolin in coating buffer. After overnight incubation, wells areblocked with 0.1% human serum albumin (HSA) in TBS (10 mM Tris-HCl, 140mM NaCl, pH 7.4), then are washed with TBS containing 0.05% Tween 20 and5 mM CaCl₂) (wash buffer). Human serum samples are diluted in 20 mMTris-HCl, 1 M NaCl, 10 mM CaCl₂), 0.05% Triton X-100, 0.1% HSA, pH 7.4,which prevents activation of endogenous C4 and dissociates the C1complex (composed of C1q, C1r and C1s). MASP-2 inhibitory agents,including anti-MASP-2 MoAbs and inhibitory peptides are added to theserum samples in varying concentrations. The diluted samples are addedto the plate and incubated overnight at 4° C. After 24 hours, the platesare washed thoroughly with wash buffer, then 0.1 μg of purified human C4(obtained as described in Dodds, A. W., Methods Enzymol. 223:46, 1993)in 100 μl of 4 mM barbital, 145 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4is added to each well. After 1.5 h at 37° C., the plates are washedagain and C4b deposition is detected using alkalinephosphatase-conjugated chicken anti-human C4c (obtained fromImmunsystem, Uppsala, Sweden) and measured using the colorimetricsubstrate p-nitrophenyl phosphate.

C4 Assay on Mannan:

The assay described above is adapted to measure lectin pathwayactivation via MBL by coating the plate with LSP and mannan prior toadding serum mixed with various MASP-2 inhibitory agents.

C4 Assay on H-Ficolin (Hakata Ag):

The assay described above is adapted to measure lectin pathwayactivation via H-ficolin by coating the plate with LPS and H-ficolinprior to adding serum mixed with various MASP-2 inhibitory agents.

Example 8

The following assay demonstrates the presence of classical pathwayactivation in wild-type and MASP-2−/− mice.

Methods:

Immune complexes were generated in situ by coating microtiter plates(Maxisorb, Nunc, cat. No. 442404, Fisher Scientific) with 0.1% humanserum albumin in 10 mM Tris, 140 mM NaCl, pH 7.4 for 1 hours at roomtemperature followed by overnight incubation at 4° C. with sheep antiwhole serum antiserum (Scottish Antibody Production Unit, Carluke,Scotland) diluted 1:1000 in TBS/tween/Ca²⁺. Serum samples were obtainedfrom wild-type and MASP-2−/− mice and added to the coated plates.Control samples were prepared in which C1q was depleted from wild-typeand MASP-2−/− serum samples. Clq-depleted mouse serum was prepared usingprotein-A-coupled Dynabeads (Dynal Biotech, Oslo, Norway) coated withrabbit anti-human C1q IgG (Dako, Glostrup, Denmark), according to thesupplier's instructions. The plates were incubated for 90 minutes at 37°C. Bound C3b was detected with a polyclonal anti-human-C3c Antibody(Dako A 062) diluted in TBS/tw/Ca⁺⁺ at 1:1000. The secondary antibody isgoat anti-rabbit IgG.

Results:

FIG. 7 shows the relative C3b deposition levels on plates coated withIgG in wild-type serum, MASP-2−/− serum, Clq-depleted wild-type andClq-depleted MASP-2−/− serum. These results demonstrate that theclassical pathway is intact in the MASP-2−/− mouse strain.

Example 9

The following assay is used to test whether a MASP-2 inhibitory agentblocks the classical pathway by analyzing the effect of a MASP-2inhibitory agent under conditions in which the classical pathway isinitiated by immune complexes.

Methods:

To test the effect of a MASP-2 inhibitory agent on conditions ofcomplement activation where the classical pathway is initiated by immunecomplexes, triplicate 50 μl samples containing 90% NHS are incubated at37° C. in the presence of 10 μg/ml immune complex (IC) or PBS, andparallel triplicate samples (+/−IC) are also included which contain 200nM anti-properdin monoclonal antibody during the 37° C. incubation.After a two hour incubation at 37° C., 13 mM EDTA is added to allsamples to stop further complement activation and the samples areimmediately cooled to 5° C. The samples are then stored at −70° C. priorto being assayed for complement activation products (C3a and sC5b-9)using ELISA kits (Quidel, Catalog Nos. A015 and A009) following themanufacturer's instructions.

Example 10

This example describes the identification of high affinity anti-MASP-2Fab2 antibody fragments that block MASP-2 activity.

Background and Rationale:

MASP-2 is a complex protein with many separate functional domains,including: binding site(s) for MBL and ficolins, a serine proteasecatalytic site, a binding site for proteolytic substrate C2, a bindingsite for proteolytic substrate C4, a MASP-2 cleavage site forautoactivation of MASP-2 zymogen, and two Ca⁺ binding sites. Fab2antibody fragments were identified that bind with high affinity toMASP-2, and the identified Fab2 fragments were tested in a functionalassay to determine if they were able to block MASP-2 functionalactivity.

To block MASP-2 functional activity, an antibody or Fab2 antibodyfragment must bind and interfere with a structural epitope on MASP-2that is required for MASP-2 functional activity. Therefore, many or allof the high affinity binding anti-MASP-2 Fab2s may not inhibit MASP-2functional activity unless they bind to structural epitopes on MASP-2that are directly involved in MASP-2 functional activity.

A functional assay that measures inhibition of lectin pathway C3convertase formation was used to evaluate the “blocking activity” ofanti-MASP-2 Fab2s. It is known that the primary physiological role ofMASP-2 in the lectin pathway is to generate the next functionalcomponent of the lectin-mediated complement pathway, namely the lectinpathway C3 convertase. The lectin pathway C3 convertase is a criticalenzymatic complex (C4bC2a) that proteolytically cleaves C3 into C3a andC3b. MASP-2 is not a structural component of the lectin pathway C3convertase (C4bC2a); however, MASP-2 functional activity is required inorder to generate the two protein components (C4b, C2a) that comprisethe lectin pathway C3 convertase. Furthermore, all of the separatefunctional activities of MASP-2 listed above appear to be required inorder for MASP-2 to generate the lectin pathway C3 convertase. For thesereasons, a preferred assay to use in evaluating the “blocking activity”of anti-MASP-2 Fab2s is believed to be a functional assay that measuresinhibition of lectin pathway C3 convertase formation.

Generation of High Affinity Fab2s:

A phage display library of human variable light and heavy chain antibodysequences and automated antibody selection technology for identifyingFab2s that react with selected ligands of interest was used to createhigh affinity Fab2s to rat MASP-2 protein (SEQ ID NO:55). A known amountof rat MASP-2 (˜1 mg, >85% pure) protein was utilized for antibodyscreening. Three rounds of amplification were utilized for selection ofthe antibodies with the best affinity. Approximately 250 different hitsexpressing antibody fragments were picked for ELISA screening. Highaffinity hits were subsequently sequenced to determine uniqueness of thedifferent antibodies.

Fifty unique anti-MASP-2 antibodies were purified and 250 μg of eachpurified Fab2 antibody was used for characterization of MASP-2 bindingaffinity and complement pathway functional testing, as described in moredetail below.

Assays used to Evaluate the Inhibitory (blocking) Activity ofAnti-MASP-2 Fab2s

1. Assay to Measure Inhibition of Formation of Lectin Pathway C3Convertase:

Background: The lectin pathway C3 convertase is the enzymatic complex(C4bC2a) that proteolytically cleaves C3 into the two potentproinflammatory fragments, anaphylatoxin C3a and opsonic C3b. Formationof C3 convertase appears to a key step in the lectin pathway in terms ofmediating inflammation. MASP-2 is not a structural component of thelectin pathway C3 convertase (C4bC2a); therefore anti-MASP-2 antibodies(or Fab2) will not directly inhibit activity of preexisting C3convertase. However, MASP-2 serine protease activity is required inorder to generate the two protein components (C4b, C2a) that comprisethe lectin pathway C3 convertase. Therefore, anti-MASP-2 Fab2 whichinhibit MASP-2 functional activity (i.e., blocking anti-MASP-2 Fab2)will inhibit de novo formation of lectin pathway C3 convertase. C3contains an unusual and highly reactive thioester group as part of itsstructure. Upon cleavage of C3 by C3 convertase in this assay, thethioester group on C3b can form a covalent bond with hydroxyl or aminogroups on macromolecules immobilized on the bottom of the plastic wellsvia ester or amide linkages, thus facilitating detection of C3b in theELISA assay.

Yeast mannan is a known activator of the lectin pathway. In thefollowing method to measure formation of C3 convertase, plastic wellscoated with mannan were incubated for 30 min at 37° C. with diluted ratserum to activate the lectin pathway. The wells were then washed andassayed for C3b immobilized onto the wells using standard ELISA methods.The amount of C3b generated in this assay is a direct reflection of thede novo formation of lectin pathway C3 convertase. Anti-MASP-2 Fab2s atselected concentrations were tested in this assay for their ability toinhibit C3 convertase formation and consequent C3b generation.

Methods:

96-well Costar Medium Binding plates were incubated overnight at 5° C.with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1 ug/50Tl/well. After overnight incubation, each well was washed three timeswith 200 Tl PBS. The wells were then blocked with 100 Tl/well of 1%bovine serum albumin in PBS and incubated for one hour at roomtemperature with gentle mixing. Each well was then washed three timeswith 200 Tl of PBS. The anti-MASP-2 Fab2 samples were diluted toselected concentrations in Ca⁺⁺ and Mg⁺⁺ containing GVB buffer (4.0 mMbarbital, 141 mM NaCl, 1.0 mM MgCl₂, 2.0 mM CaCl₂, 0.1% gelatin, pH 7.4)at 5 C. A 0.5% rat serum was added to the above samples at 5 C and 100Tl was transferred to each well. Plates were covered and incubated for30 minutes in a 37 C waterbath to allow complement activation. Thereaction was stopped by transferring the plates from the 37 C waterbathto a container containing an ice-water mix. Each well was washed fivetimes with 200 Tl with PBS-Tween 20 (0.05% Tween 20 in PBS), then washedtwo times with 200 Tl PBS. A 100 Tl/well of 1:10,000 dilution of theprimary antibody (rabbit anti-human C3c, DAKO A0062) was added in PBScontaining 2.0 mg/ml bovine serum albumin and incubated 1 hr at roomtemperature with gentle mixing. Each well was washed 5×200 Tl PBS. 100Tl/well of 1:10,000 dilution of the secondary antibody(peroxidase-conjugated goat anti-rabbit IgG, American Qualex A102PU) wasadded in PBS containing 2.0 mg/ml bovine serum albumin and incubated forone hour at room temperature on a shaker with gentle mixing. Each wellwas washed five times with 200 Tl with PBS. 100 Tl/well of theperoxidase substrate TMB (Kirkegaard & Perry Laboratories) was added andincubated at room temperature for 10 min. The peroxidase reaction wasstopped by adding 100 Ti/well of 1.0 M H₃PO₄ and the OD₄₅₀. wasmeasured.

2. Assay to Measure Inhibition of MASP-2-Dependent C4 Cleavage

Background: The serine protease activity of MASP-2 is highly specificand only two protein substrates for MASP-2 have been identified; C2 andC4. Cleavage of C4 generates C4a and C4b. Anti-MASP-2 Fab2 may bind tostructural epitopes on MASP-2 that are directly involved in C4 cleavage(e.g., MASP-2 binding site for C4; MASP-2 serine protease catalyticsite) and thereby inhibit the C4 cleavage functional activity of MASP-2.

Yeast mannan is a known activator of the lectin pathway. In thefollowing method to measure the C4 cleavage activity of MASP-2, plasticwells coated with mannan were incubated for 30 minutes at 37 C withdiluted rat serum to activate the lectin pathway. Since the primaryantibody used in this ELISA assay only recognizes human C4, the dilutedrat serum was also supplemented with human C4 (1.0 Tg/ml). The wellswere then washed and assayed for human C4b immobilized onto the wellsusing standard ELISA methods. The amount of C4b generated in this assayis a measure of MASP-2 dependent C4 cleavage activity. Anti-MASP-2 Fab2at selected concentrations were tested in this assay for their abilityto inhibit C4 cleavage.

Methods:

96-well Costar Medium Binding plates were incubated overnight at 5 Cwith mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1.0 Tg/50Tl/well. Each well was washed 3× with 200 Ti PBS. The wells were thenblocked with 100 Tl/well of 1% bovine serum albumin in PBS and incubatedfor one hour at room temperature with gentle mixing. Each well waswashed 3× with 200 Tl of PBS. Anti-MASP-2 Fab2 samples were diluted toselected concentrations in Ca⁺⁺ and Mg⁺⁺ containing GVB buffer (4.0 mMbarbital, 141 mM NaCl, 1.0 mM MgCl₂, 2.0 mM CaCl₂, 0.1% gelatin, pH 7.4)at 5 C. 1.0 Tg/ml human C4 (Quidel) was also included in these samples.0.5% rat serum was added to the above samples at 5 C and 100 Ti wastransferred to each well. The plates were covered and incubated for 30min in a 37 C waterbath to allow complement activation. The reaction wasstopped by transferring the plates from the 37 C waterbath to acontainer containing an ice-water mix. Each well was washed 5×200 Tiwith PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washedwith 2× with 200 Tl PBS. 100 Tl/well of 1:700 dilution ofbiotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala,Sweden) was added in PBS containing 2.0 mg/ml bovine serum albumin (BSA)and incubated one hour at room temperature with gentle mixing. Each wellwas washed 5×200 Ti PBS. 100 Tl/well of 0.1 Tg/ml ofperoxidase-conjugated streptavidin (Pierce Chemical #21126) was added inPBS containing 2.0 mg/ml BSA and incubated for one hour at roomtemperature on a shaker with gentle mixing. Each well was washed 5×200Tl with PBS. 100 Tl/well of the peroxidase substrate TMB (Kirkegaard &Perry Laboratories) was added and incubated at room temperature for 16min. The peroxidase reaction was stopped by adding 100 Tl/well of 1.0 MH₃PO₄ and the OD₄₅₀ was measured.

3. Binding Assay of Anti-Rat MASP-2 Fab2 to ‘Native’ Rat MASP-2

Background: MASP-2 is usually present in plasma as a MASP-2 dimercomplex that also includes specific lectin molecules (mannose-bindingprotein (MBL) and ficolins). Therefore, if one is interested in studyingthe binding of anti-MASP-2 Fab2 to the physiologically relevant form ofMASP-2, it is important to develop a binding assay in which theinteraction between the Fab2 and ‘native’ MASP-2 in plasma is used,rather than purified recombinant MASP-2. In this binding assay the‘native’ MASP-2-MBL complex from 10% rat serum was first immobilizedonto mannan-coated wells. The binding affinity of various anti-MASP-2Fab2s to the immobilized ‘native’ MASP-2 was then studied using astandard ELISA methodology.

Methods:

96-well Costar High Binding plates were incubated overnight at 5° C.with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1 Tg/50Tl/well. Each well was washed 3× with 200 Tl PBS. The wells were blockedwith 100 Tl/well of 0.5% nonfat dry milk in PBST (PBS with 0.05% Tween20) and incubated for one hour at room temperature with gentle mixing.Each well was washed 3× with 200 Tl of TBS/Tween/Ca Wash Buffer(Tris-buffered saline, 0.05% Tween 20, containing 5.0 mM CaCl₂, pH 7.4.10% rat serum in High Salt Binding Buffer (20 mM Tris, 1.0 M NaCl, 10 mMCaCl₂, 0.05% Triton-X100, 0.1% (w/v) bovine serum albumin, pH 7.4) wasprepared on ice. 100 Tl/well was added and incubated overnight at 5° C.Wells were washed 3× with 200 Tl of TBS/Tween/Ca⁺⁺ Wash Buffer. Wellswere then washed 2× with 200 Tl PBS. 100 Tl/well of selectedconcentration of anti-MASP-2 Fab2 diluted in Ca⁺⁺ and Mg⁺⁺ containingGVB Buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl₂, 2.0 mM CaCl₂,0.1% gelatin, pH 7.4) was added and incubated for one hour at roomtemperature with gentle mixing. Each well was washed 5×200 Tl PBS. 100Tl/well of HRP-conjugated goat anti-Fab2 (Biogenesis Cat No 0500-0099)diluted 1:5000 in 2.0 mg/ml bovine serum albumin in PBS was added andincubated for one hour at room temperature with gentle mixing. Each wellwas washed 5×200 Tl PBS. 100 Tl/well of the peroxidase substrate TMB(Kirkegaard & Perry Laboratories) was added and incubated at roomtemperature for 70 min. The peroxidase reaction was stopped by adding100 Ti/well of 1.0 M H₃PO₄ and OD₄₅₀. was measured.

Results:

Approximately 250 different Fab2s that reacted with high affinity to therat MASP-2 protein were picked for ELISA screening. These high affinityFab2s were sequenced to determine the uniqueness of the differentantibodies, and 50 unique anti-MASP-2 antibodies were purified forfurther analysis. 250 ug of each purified Fab2 antibody was used forcharacterization of MASP-2 binding affinity and complement pathwayfunctional testing. The results of this analysis is shown below in TABLE6.

TABLE 6 ANTI-MASP-2 FAB2 THAT BLOCK LECTIN PATHWAY COMPLEMENT ACTIVATIONFab2 C3 Convertase C4 Cleavage antibody # (IC₅₀ (nM) K_(d) IC₅₀ (nM) 880.32 4.1 ND 41 0.35 0.30 0.81 11 0.46 0.86 <2 nM 86 0.53 1.4 ND 81 0.542.0 ND 66 0.92 4.5 ND 57 0.95 3.6 <2 nM 40 1.1 7.2 0.68 58 1.3 2.6 ND 601.6 3.1 ND 52 1.6 5.8 <2 nM 63 2.0 6.6 ND 49 2.8 8.5 <2 nM 89 3.0 2.5 ND71 3.0 10.5 ND 87 6.0 2.5 ND 67 10.0 7.7 ND

As shown above in TABLE 6, of the 50 anti-MASP-2 Fab2s tested, seventeenFab2s were identified as MASP-2 blocking Fab2 that potently inhibit C3convertase formation with IC₅₀ equal to or less than 10 nM Fab2s (a 34%positive hit rate). Eight of the seventeen Fab2s identified have IC₅₀sin the subnanomolar range. Furthermore, all seventeen of the MASP-2blocking Fab2s shown in TABLE 6 gave essentially complete inhibition ofC3 convertase formation in the lectin pathway C3 convertase assay. FIG.8A graphically illustrates the results of the C3 convertase formationassay for Fab2 antibody #11, which is representative of the other Fab2antibodies tested, the results of which are shown in TABLE 6. This is animportant consideration, since it is theoretically possible that a“blocking” Fab2 may only fractionally inhibit MASP-2 function even wheneach MASP-2 molecule is bound by the Fab2.

Although mannan is a known activator of the lectin pathway, it istheoretically possible that the presence of anti-mannan antibodies inthe rat serum might also activate the classical pathway and generate C3bvia the classical pathway C3 convertase. However, each of the seventeenblocking anti-MASP-2 Fab2s listed in this example potently inhibits C3bgeneration (>95%), thus demonstrating the specificity of this assay forlectin pathway C3 convertase.

Binding assays were also performed with all seventeen of the blockingFab2s in order to calculate an apparent K_(d) for each. The results ofthe binding assays of anti-rat MASP-2 Fab2s to native rat MASP-2 for sixof the blocking Fab2s are also shown in TABLE 6.

FIG. 8B graphically illustrates the results of a binding assay with theFab2 antibody #11. Similar binding assays were also carried out for theother Fab2s, the results of which are shown in TABLE 6. In general, theapparent Kds obtained for binding of each of the six Fab2s to ‘native’MASP-2 corresponds reasonably well with the IC₅₀ for the Fab2 in the C3convertase functional assay. There is evidence that MASP-2 undergoes aconformational change from an ‘inactive’ to an ‘active’ form uponactivation of its protease activity (Feinberg et al., EMBO J 22:2348-59(2003); Gal et al., J. Biol. Chem. 280:33435-44 (2005)). In the normalrat plasma used in the C3 convertase formation assay, MASP-2 is presentprimarily in the ‘inactive’ zymogen conformation. In contrast, in thebinding assay, MASP-2 is present as part of a complex with MBL bound toimmobilized mannan; therefore, the MASP-2 would be in the ‘active’conformation (Petersen et al., J. Immunol Methods 257:107-16, 2001).Consequently, one would not necessarily expect an exact correspondencebetween the IC₅₀ and K_(d) for each of the seventeen blocking Fab2tested in these two functional assays since in each assay the Fab2 wouldbe binding a different conformational form of MASP-2. Never-the-less,with the exception of Fab2 #88, there appears to be a reasonably closecorrespondence between the IC₅₀ and apparent Kd for each of the othersixteen Fab2 tested in the two assays (see TABLE 6).

Several of the blocking Fab2s were evaluated for inhibition of MASP-2mediated cleavage of C4. FIG. 8C graphically illustrates the results ofa C4 cleavage assay, showing inhibition with Fab2 #41, with an IC₅₀=0.81nM (see TABLE 6). As shown in FIG. 9, all of the Fab2s tested were foundto inhibit C4 cleavage with IC₅₀s similar to those obtained in the C3convertase assay (see TABLE 6).

Although mannan is a known activator of the lectin pathway, it istheoretically possible that the presence of anti-mannan antibodies inthe rat serum might also activate the classical pathway and therebygenerate C4b by C1s-mediated cleavage of C4. However, severalanti-MASP-2 Fab2s have been identified which potently inhibit C4bgeneration (>95%), thus demonstrating the specificity of this assay forMASP-2 mediated C4 cleavage. C4, like C3, contains an unusual and highlyreactive thioester group as part of its structure. Upon cleavage of C4by MASP-2 in this assay, the thioester group on C4b can form a covalentbond with hydroxyl or amino groups on macromolecules immobilized on thebottom of the plastic wells via ester or amide linkages, thusfacilitating detection of C4b in the ELISA assay.

These studies clearly demonstrate the creation of high affinity FAB2s torat MASP-2 protein that functionally block both C4 and C3 convertaseactivity, thereby preventing lectin pathway activation.

Example 11

This Example describes the epitope mapping for several of the blockinganti-rat MASP-2 Fab2 antibodies that were generated as described inExample 10.

Methods:

As shown in FIG. 10, the following proteins, all with N-terminal 6× Histags were expressed in CHO cells using the pED4 vector:

rat MASP-2A, a full length MASP-2 protein, inactivated by altering theserine at the active center to alanine (S613A);

rat MASP-2K, a full-length MASP-2 protein altered to reduceautoactivation (R424K);

CUBI-II, an N-terminal fragment of rat MASP-2 that contains the CUBI,EGF-like and CUBII domains only; and

CUBI/EGF-like, an N-terminal fragment of rat MASP-2 that contains theCUBI and EGF-like domains only.

These proteins were purified from culture supernatants bynickel-affinity chromatography, as previously described (Chen et al., J.Biol. Chem. 276:25894-02 (2001)).

A C-terminal polypeptide (CCPII-SP), containing CCPII and the serineprotease domain of rat MASP-2, was expressed in E. coli as a thioredoxinfusion protein using pTrxFus (Invitrogen). Protein was purified fromcell lysates using Thiobond affinity resin. The thioredoxin fusionpartner was expressed from empty pTrxFus as a negative control.

All recombinant proteins were dialyzed into TBS buffer and theirconcentrations determined by measuring the OD at 280 nm.

Dot Blot Analysis:

Serial dilutions of the five recombinant MASP-2 polypeptides describedabove and shown in FIG. 10 (and the thioredoxin polypeptide as anegative control for CCPII-serine protease polypeptide) were spottedonto a nitrocellulose membrane. The amount of protein spotted rangedfrom 100 ng to 6.4 pg, in five-fold steps. In later experiments, theamount of protein spotted ranged from 50 ng down to 16 pg, again infive-fold steps. Membranes were blocked with 5% skimmed milk powder inTBS (blocking buffer) then incubated with 1.0 μg/ml anti-MASP-2 Fab2s inblocking buffer (containing 5.0 mM Ca²⁺). Bound Fab2s were detectedusing HRP-conjugated anti-human Fab (AbD/Serotec; diluted 1/10,000) andan ECL detection kit (Amersham). One membrane was incubated withpolyclonal rabbit-anti human MASP-2 Ab (described in Stover et al., JImmunol 163:6848-59 (1999)) as a positive control. In this case, boundAb was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted1/2,000).

MASP-2 Binding Assay

ELISA plates were coated with 1.0 gpg/well of recombinant MASP-2A orCUBI-II polypeptide in carbonate buffer (pH 9.0) overnight at 4° C.Wells were blocked with 1% BSA in TBS, then serial dilutions of theanti-MASP-2 Fab2s were added in TBS containing 5.0 mM Ca²′. The plateswere incubated for one hour at RT. After washing three times withTBS/tween/Ca²⁺, HRP-conjugated anti-human Fab (AbD/Serotec) diluted1/10,000 in TBS/Ca²⁺ was added and the plates incubated for a furtherone hour at RT. Bound antibody was detected using a TMB peroxidasesubstrate kit (Biorad).

Results:

Results of the dot blot analysis demonstrating the reactivity of theFab2s with various MASP-2 polypeptides are provided below in TABLE 7.The numerical values provided in TABLE 7 indicate the amount of spottedprotein required to give approximately half-maximal signal strength. Asshown, all of the polypeptides (with the exception of the thioredoxinfusion partner alone) were recognized by the positive control Ab(polyclonal anti-human MASP-2 sera, raised in rabbits).

TABLE 7 REACTIVITY WITH VARIOUS RECOMBINANT RAT MASP-2 POLYPEPTIDES ONDOT BLOTS Fab2 MASP- CUBI- CUBI/EGF- CCPII- Thiore- Antibody # 2A IIlike SP doxin 40 0.16 ng NR NR 0.8 ng NR 41 0.16 ng NR NR 0.8 ng NR 110.16 ng NR NR 0.8 ng NR 49 0.16 ng NR NR >20 ng NR 52 0.16 ng NR NR 0.8ng NR 57 0.032 ng NR NR NR NR 58 0.4 ng NR NR 2.0 ng NR 60 0.4 ng  0.4ng NR NR NR 63 0.4 ng NR NR 2.0 ng NR 66 0.4 ng NR NR 2.0 ng NR 67 0.4ng NR NR 2.0 ng NR 71 0.4 ng NR NR 2.0 ng NR 81 0.4 ng NR NR 2.0 ng NR86 0.4 ng NR NR 10 ng NR 87 0.4 ng NR NR 2.0 ng NR Positive <0.032 ng0.16 ng 0.16 ng <0.032 ng NR Controlpathway activity was observed over the second and third weeks, withcomplete lectin pathway restoration in the mice by 17 days postanti-MASP-2 MoAb administration. NR=No reaction. The positive controlantibody is polyclonal anti-human MASP-2 sera, raised in rabbits.

All of the Fab2s reacted with MASP-2A as well as MASP-2K (data notshown). The majority of the Fab2s recognized the CCPII-SP polypeptidebut not the N-terminal fragments. The two exceptions are Fab2 #60 andFab2 #57. Fab2 #60 recognizes MASP-2A and the CUBI-II fragment, but notthe CUBI/EGF-like polypeptide or the CCPII-SP polypeptide, suggesting itbinds to an epitope in CUBII, or spanning the CUBII and the EGF-likedomain. Fab2 #57 recognizes MASP-2A but not any of the MASP-2 fragmentstested, indicating that this Fab2 recognizes an epitope in CCP1. Fab2#40 and #49 bound only to complete MASP-2A. In the ELISA binding assayshown in FIG. 11, Fab2 #60 also bound to the CUBI-II polypeptide, albeitwith a slightly lower apparent affinity.

These finding demonstrate the identification of unique blocking Fab2s tomultiple regions of the MASP-2 protein

Example 12

This Example describes the analysis of MASP-2−/− mice in a Murine RenalIschemia/Reperfusion Model.

Background/Rationale:

Ischemia-Reperfusion (I/R) injury in kidney at body temperature hasrelevance in a number of clinical conditions, including hypovolaemicshock, renal artery occlusion and cross-clamping procedures.

Kidney ischemia-reperfusion (I/R) is an important cause of acute renalfailure, associated with a mortality rate of up to 50% (Levy et al.,JAMA 275:1489-94, 1996; Thadhani et al., N. Engl. J. Med. 334:1448-60,1996). Post-transplant renal failure is a common and threateningcomplication after renal transplantation (Nicholson et al., Kidney Int.58:2585-91, 2000). Effective treatment for renal YR injury is currentlynot available and hemodialysis is the only treatment available. Thepathophysiology of renal YR injury is complicated. Recent studies haveshown that the lectin pathway of complement activation may have animportant role in the pathogenesis of renal IR injury (deVries et al.,Am. J. Path. 165:1677-88, 2004).

Methods:

A MASP-2(−/−) mouse was generated as described in Example 1 andbackcrossed for at least 10 generations with C57B1/6. Six maleMASP-2(−/−) and six wildtype (+/+) mice weighing between 22-25 g wereadministered an intraperitoneal injection of Hypnovel (6.64 mg/kg; Rocheproducts Ltd. Welwyn Garden City, UK), and subsequently anaesthetized byinhalation of isoflurane (Abbott Laboratories Ltd., Kent, UK).Isoflurane was chosen because it is a mild inhalation anaesthetic withminimal liver toxicity; the concentrations are produced accurately andthe animal recovers rapidly, even after prolonged anaesthesia. Hypnovelwas administered because it produces a condition of neuroleptanalgesiain the animal and means that less isoflurane needs to be administered. Awarm pad was placed beneath the animal in order to maintain a constantbody temperature. Next, a midline abdominal incision was performed andthe body cavity held open using a pair of retractors. Connective tissuewas cleared above and below the renal vein and artery of both right andleft kidneys, and the renal pedicle was clamped via the application ofmicroaneurysm clamps for a period of 55 minutes. This period of ischemiawas based initially on a previous study performed in this laboratory(Zhou et al., J. Clin. Invest. 105:1363-71 (2000)). In addition, astandard ischemic time of 55 minutes was chosen following ischemictitration and it was found that 55 minutes gave consistent injury thatwas also reversible, with low mortality, less than 5%. After occlusion,0.4 ml of warm saline (37° C.) was placed in the abdominal cavity andthen the abdomen was closed for the period of ischemia. Followingremoval of the microaneurysm clamps, the kidneys were observed untilcolor change, an indication of blood re-flow to the kidneys. A further0.4 ml of warm saline was placed in the abdominal cavity and the openingwas sutured, whereupon animals were returned to their cages. Tail bloodsamples were taken at 24 hours after removing the clamps, and at 48hours the mice were sacrificed and an additional blood sample wascollected.

Assessment of Renal Injury:

Renal function was assessed at 24 and 48 hours after reperfusion in sixmale MASP-2(−/−) and six WT (+/+) mice. Blood creatinine measurement wasdetermined by mass spectrometry, which provides a reproducible index ofrenal function (sensitivity <1.0 μmol/L). FIG. 12 graphicallyillustrates the blood urea nitrogen clearance for wildtype C57Bl/6controls and MASP-2 (−/−) at 24 hours and 48 hours after reperfusion. Asshown in FIG. 12, MASP-2(−/−) mice displayed a significant reduction inthe amount of blood urea at 24 and 48 hours, in comparison to wildtypecontrol mice, indicating a protective functional effect from renaldamage in the ischemia reperfusion injury model.

Overall, increased blood urea was seen in both the WT (+/+) and MASP-2(−/−) mice at 24 and 48 hours following the surgical procedure andischemic insult. Levels of blood urea in a non-ischemic WT (+/+) surgeryanimal was separately determined to be 5.8 mmol/L. In addition to thedata presented in FIG. 12, one MASP-2 (−/−) animal showed nearlycomplete protection from the ischemic insult, with values of 6.8 and 9.6mmol/L at 24 and 48 hours, respectively. This animal was excluded fromthe group analysis as a potential outlier, wherein no ischemic injurymay have been present. Therefore, the final analysis shown in FIG. 12included 5 MASP-2(−/−) mice and 6 WT (+/+) mice and a statisticallysignificant reduction in blood urea was seen at 24 and 48 hours in theMASP-2 (−/−) mice (Student t-test p<0.05). These findings indicateinhibition of MASP-2 activity would be expected to have a protective ortherapeutic effect from renal damage due to ischemic injury.

Example 13

This Example describes the results of MASP-2−/− in a Murine MacularDegeneration Model.

Background/Rationale:

Age-related macular degeneration (AMD) is the leading cause of blindnessafter age 55 in the industrialized world. AMD occurs in two major forms:neovascular (wet) AMD and atrophic (dry) AMD. The neovascular (wet) formaccounts for 90% of severe visual loss associated with AMD, even thoughonly ˜20% of individuals with AMD develop the wet form. Clinicalhallmarks of AMD include multiple drusen, geographic atrophy, andchoroidal neovascularization (CNV). In December, 2004, the FDA approvedMacugen (pegaptanib), a new class of ophthalmic drugs to specificallytarget and block the effects of vascular endothelial growth factor(VEGF), for treatment of the wet (neovascular) form of AMD (Ng et al.,Nat Rev. Drug Discov 5:123-32 (2006)). Although Macugen represents apromising new therapeutic option for a subgroup of AMD patients, thereremains a pressing need to develop additional treatments for thiscomplex disease. Multiple, independent lines of investigation implicatea central role for complement activation in the pathogenesis of AMD. Thepathogenesis of choroidal neovascularization (CNV), the most seriousform of AMD, may involve activation of complement pathways.

Over twenty-five years ago, Ryan described a laser-induced injury modelof CNV in animals (Ryan, S. J., Tr. Am. Opth. Soc. LXXVII:707-745,1979). The model was initially developed using rhesus monkeys, however,the same technology has since been used to develop similar models of CNVin a variety of research animals, including the mouse (Tobe et al., Am.J. Pathol. 153:1641-46, 1998). In this model, laser photocoagulation isused to break Bruch's membrane, an act which results in the formation ofCNV-like membranes. The laser-induced model captures many of theimportant features of the human condition (for a recent review, seeAmbati et al., Survey Ophthalmology 48:257-293, 2003). The laser-inducedmouse model is now well established, and is used as an experimentalbasis in a large, and ever increasing, number of research projects. Itis generally accepted that the laser-induced model shares enoughbiological similarity with CNV in humans that preclinical studies ofpathogenesis and drug inhibition using this model are relevant to CNV inhumans.

Methods:

A MASP-2−/− mouse was generated as described in Example 1 andbackcrossed for 10 generations with C57Bl/6. The current study comparedthe results when MASP-2 (−/−) and MASP-2 (+/+) male mice were evaluatedin the course of laser-induced CNV, an accelerated model of neovascularAMD focusing on the volume of laser-induced CNV by scanning laserconfocal microscopy as a measure of tissue injury and determination oflevels of VEGF, a potent angiogenic factor implicated in CNV, in theretinal pigment epithelium (RPE)/choroids by ELISA after laser injury.

Induction of Choroidal Neovascularization (CNV):

Laser photocoagulation (532 nm, 200 mW, 100 ms, 75 μm; Oculight GL,Iridex, Mountain View, Calif.) was performed on both eyes of each animalon day zero by a single individual masked to drug group assignment.Laser spots were applied in a standardized fashion around the opticnerve, using a slit lamp delivery system and a coverslip as a contactlens. The morphologic end point of the laser injury was the appearanceof a cavitation bubble, a sign thought to correlate with the disruptionof Bruch's membrane. The detailed methods and endpoints that wereevaluated are as follows.

Fluorescein Angiography:

Fluorescein angiography was performed with a camera and imaging system(TRC 50 1A camera; ImageNet 2.01 system; Topcon, Paramus, N.J.) at 1week after laser photocoagulation. The photographs were captured with a20-D lens in contact with the fundus camera lens after intraperitonealinjection of 0.1 ml of 2.5% fluorescein sodium. A retina expert notinvolved in the laser photocoagulation or angiography evaluated thefluorescein angiograms at a single sitting in masked fashion.

Volume of Choroidal Neovascularization (CNV):

One week after laser injury, eyes were enucleated and fixed with 4%paraformaldehyde for 30 min at 4° C. Eye cups were obtained by removinganterior segments and were washed three times in PBS, followed bydehydration and rehydration through a methanol series. After blockingtwice with buffer (PBS containing 1% bovine serumalbumin and 0.5% TritonX-100) for 30 minutes at room temperature, eye cups were incubatedovernight at 4° C. with 0.5% FITC-isolectin B4 (Vector laboratories,Burlingame, Calif.), diluted with PBS containing 0.2% BSA and 0.1%Triton X-100, which binds terminal j3-D-galactose residues on thesurface of endothelial cells and selectively labels the murinevasculature. After two washings with PBS containing 0.1% Triton X-100,the neurosensory retina was gently detached and severed from the opticnerve. Four relaxing radial incisions were made, and the remainingRPE-choroid-sclera complex was flatmounted in antifade medium(Immu-Mount Vectashield Mounting Medium; Vector Laboratories) andcover-slipped.

Flatmounts were examined with a scanning laser confocal microscope (TCSSP; Leica, Heidelberg, Germany). Vessels were visualized by excitingwith blue argon wavelength (488 nm) and capturing emission between 515and 545 nm. A 40× oil-immersion objective was used for all imagingstudies. Horizontal optical sections (1 pm step) were obtained from thesurface of the RPE-choroid-sclera complex. The deepest focal plane inwhich the surrounding choroidal vascular network connecting to thelesion could be identified was judged to be the floor of the lesion. Anyvessel in the laser-targeted area and superficial to this referenceplane was judged as CNV. Images of each section were digitally stored.The area of CNV-related fluorescence was measured by computerized imageanalysis with the microscope software (TCS SP; Leica). The summation ofwhole fluorescent area in each horizontal section was used as an indexfor the volume of CNV. Imaging was performed by an operator masked totreatment group assignment.

Because the probability of each laser lesion developing CNV isinfluenced by the group to which it belongs (mouse, eye, and laserspot), the mean lesion volumes were compared using a linear mixed modelwith a split plot repeated-measures design. The whole plot factor wasthe genetic group to which the animal belongs, whereas the split plotfactor was the eye. Statistical significance was determined at the 0.05level. Post hoc comparisons of means were constructed with a Bonferroniadjustment for multiple comparisons.

VEGF ELISA.

At three days after injury by 12 laser spots, the RPE-choroid complexwas sonicated in lysis buffer (20 mM imidazole HCl, 10 mM KCl, 1 mMMgCL₂, 10 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate, and 1mM EDTA with protease inhibitor) on ice for 15 min. VEGF protein levelsin the supernatant were determined by an ELISA kit (R&D Systems,Minneapolis, Minn.) that recognizes all splice variants, at 450 to 570nm (Emax; Molecular Devices, Sunnyvale, Calif.), and normalized to totalprotein. Duplicate measurements were performed in a masked fashion by anoperator not involved in photocoagulation, imaging, or angiography. VEGFnumbers were represented as the mean+/−SEM of at least three independentexperiments and compared using the Mann-Whitney U test. The nullhypothesis was rejected at P<0.05.

Results:

Assessment of VEGF Levels:

FIG. 13A graphically illustrates the VEGF protein levels in RPE-choroidcomplex isolated from C57B16 wildtype and MASP-2(−/−) mice at day zero.As shown in FIG. 13A, the assessment of VEGF levels indicate a decreasein baseline levels for VEGF in the MASP-2 (−/−) mice versus the C57blwildtype control mice. FIG. 13B graphically illustrates VEGF proteinlevels measured at day three following laser induced injury. As shown inFIG. 13B VEGF levels were significantly increased in the wildtype (+/+)mice three days following laser induced injury, consistent withpublished studies (Nozaki et al., Proc. Natl. Acad. Sci. USA 103:2328-33(2006)). However, surprisingly very low levels of VEGF were seen in theMASP-2 (−/−) mice.

Assessment of Choroidal Neovascularization (CNV):

In addition to the reduction in VEGF levels following laser inducedmacular degeneration, CNV area was determined before and after laserinjury. FIG. 14 graphically illustrates the CNV volume measured in C57blwildtype mice and MASP-2(−/−) mice at day seven following laser inducedinjury. As shown in FIG. 14, the MASP-2 (−/−) mice displayed about a 30%reduction in the CNV area following laser induced damage at day seven incomparison to the wildtype control mice.

These findings indicate a reduction in VEGF and CNV as seen in the MASP(−/−) mice versus the wildtype (+/+) control and that blockade of MASP-2with an inhibitor would have a preventive or therapeutic effect in thetreatment of macular degeneration.

Example 14

This Example demonstrates that thrombin activation can occur followinglectin pathway activation under physiological conditions, anddemonstrates the extent of MASP-2 involvement. In normal rat serum,activation of the lectin pathway leads to thrombin activation (assessedas thrombin deposition) concurrent with complement activation (assessedas C4 deposition). As can be seen in FIGS. 15A and 15B, thrombinactivation in this system is inhibited by a MASP-2 blocking antibody(Fab2 format), exhibiting an inhibition concentration-response curve(FIG. 15B) that parallels that for complement activation (FIG. 15A).These data suggest that activation of the lectin pathway as it occurs intrauma will lead to activation of both complement and coagulationsystems in a process that is entirely dependent on MASP-2. By inference,MASP2 blocking antibodies may prove efficacious in mitigating cases ofexcessive systemic coagulation, e.g., disseminated intravascularcoagulation, which is one of the hallmarks leading to mortality in majortrauma cases.

Example 15

This Example provides results generated using a localized Schwartzmanreaction model of disseminated intravascular coagulation (“DIC”) inMASP-2 −/− deficient and MASP-2+/+sufficient mice to evaluate the roleof lectin pathway in DIC.

Background/Rationale:

As described supra, blockade of MASP-2 inhibits lectin pathwayactivation and reduces the generation of both anaphylatoxins C3a andC5a. C3a anaphylatoxins can be shown to be potent platelet aggregatorsin vitro, but their involvement in vivo is less well defined and therelease of platelet substances and plasmin in wound repair may onlysecondarily involve complement C3. In this Example, the role of thelectin pathway was analyzed in MASP-2 (−/−) and WT (+/+) mice in orderto address whether prolonged elevation of C3 activation is necessary togenerate disseminated intravascular coagulation.

Methods:

The MASP-2 (−/−) mice used in this study were generated as described inExample 1 and backcrossed for at least 10 generations with C57Bl/6.

The localized Schwartzman reaction model was used in this experiment.The localized Schwartzman reaction (LSR) is a lipopolysaccharide(LPS)-induced response with well-characterized contributions fromcellular and humoral elements of the innate immune system. Dependent ofthe LSR on complement is well established (Polak, L., et al., Nature223:738-739 (1969); Fong J. S. et al., J Exp Med 134:642-655 (1971)). Inthe LSR model, the mice were primed for 4 hours with TNF alpha (500 ng,intrascrotal), then the mice were anaesthetized and prepared forintravital microscopy of the cremaster muscle. Networks ofpost-capillary venules (15-60 jpm diameter) with good blood flow (1-4mm/s) were selected for observation. Animals were treated withfluorescent antibodies to selectively label neutrophils, or platelets.The network of vessels was sequentially scanned and images of allvessels were digitally recorded of later analysis. After recording thebasal state of the microcirculation, mice received a single intravenousinjection of LPS (100 μg), either alone or with the agents listed below.The same network of vessels was then scanned every 10 minutes for 1hour. Specific accumulation of fluorophores was identified bysubtraction of background fluorescence and enhanced by thresholding theimage. The magnitude of reactions was measured from recorded images. Theprimary measure of Schwartzman reactions was aggregate data.

The studies compared the MASP-2+/+sufficient, or wild type, mice exposedto either a known complement pathway depletory agent, cobra venom factor(CVF), or a terminal pathway inhibitor (C5aR antagonist). The results(FIG. 16A) demonstrate that CVF as well as a C5aR antagonist bothprevented the appearance of aggregates in the vasculature. In addition,the MASP-2 −/− deficient mice (FIG. 16B) also demonstrated completeinhibition of the localized Schwartzman reaction, supporting lectinpathway involvement. These results clearly demonstrate the role ofMASP-2 in DIC generation and support the use of MASP-2 inhibitors forthe treatment and prevention of DIC.

Example 16

This Example describes the analysis of MASP-2 (−/−) mice in a MurineRenal Transplantation Model.

Background/Rationale:

The role of MASP-2 in the functional outcome of kidney transplantationwas assessed using a mouse model.

Methods:

The functional outcome of kidney transplantation was assessed using asingle kidney isograft into uninephrecomized recipient mice, with six WT(+/+) transplant recipients (B6), and six MASP-2 (−/−) transplantrecipients. To assess the function of the transplanted kidney, theremaining native kidney was removed from the recipient 5 days aftertransplantation, and renal function was assessed 24 hours later bymeasurement of blood urea nitrogen (BUN) levels.

Results:

FIG. 17 graphically illustrates the blood urea nitrogen (BUN) levels ofthe kidney at 6 days post kidney transplant in the WT (+/+) recipientsand the MASP-2 (−/−) recipients. As shown in FIG. 17, strongly elevatedBUN levels were observed in the WT (+/+) (B6) transplant recipients(normal BUN levels in mice are <5 mM), indicating renal failure. Incontrast, MASP-2 (−/−) isograft recipient mice showed substantiallylower BUN levels, suggesting improved renal function. It is noted thatthese results were obtained using grafts from WT (+/+) kidney donors,suggesting that the absence of a functional lectin pathway in thetransplant recipient alone is sufficient to achieve a therapeuticbenefit. Taken together, these results indicate that transientinhibition of the lectin pathway via MASP-2 inhibition provides a methodof reducing morbidity and delayed graft function in renaltransplantation, and that this approach is likely to be useful in othertransplant settings.

Example 17

This Example demonstrates that MASP-2 (−/−) mice are resistant to septicshock in a Murine Polymicrobial Septic Peritonitis Model.

Background/Rationale:

To evaluate the potential effects of MASP-2 (−/−) in infection, thececal ligation and puncture (CLP) model, a model of polymicrobial septicperitonitis was evaluated. This model is thought to most accuratelymimic the course of human septic peritonitis. The cecal ligation andpuncture (CLP) model is a model in which the cecum is ligated andpunctured by a needle, leading to continuous leakage of the bacteriainto the abdominal cavity which reach the blood through the lymphdrainage and are then distributed into all the abdominal organs, leadingto multi-organ failure and septic shock (Eskandari et al., J Immunol148(9):2724-2730 (1992)). The CLP model mimics the course of sepsisobserved in patients and induces an early hyper-inflammatory responsefollowed by a pronounced hypo-inflammatory phase. During this phase, theanimals are highly sensitive to bacterial challenges (Wichterman et al.,J. Surg. Res. 29(2):189-201 (1980)).

Methods:

The mortality of polymicrobial infection using the cecal ligation andpuncture (CLP) model was measured in WT (+/+) (n=18) and MASP-2 (−/−)(n=16) mice. Briefly described, MASP-2 deficient mice and theirwild-type littermates were anaesthetized and the cecum was exteriorizedand ligated 30% above the distal end. After that, the cecum waspunctured once with a needle of 0.4 mm diameter. The cecum was thenreplaced into the abdominal cavity and the skin was closed with clamps.The survival of the mice subjected to CLP was monitored over a period of14 days after CLP. A peritoneal lavage was collected in mice 16 hourspost CLP to measure bacterial load. Serial dilutions of the peritoneallavage were prepared in PBS and inoculated in Mueller Hinton plates withsubsequent incubation at 37° C. under anaerobic conditions for 24 hoursafter which bacterial load was determined.

The TNF-alpha cytokine response to the bacterial infection was alsomeasured in the WT (+/+) and MASP-2 (−/−) mice 16 hours after CLP inlungs and spleens via quantitative real time polymerase chain reaction(qRT-PCR). The serum level of TNF-alpha 16 hours after CLP in the WT(+/+) and MASP-2 (−/−) mice was also quantified by sandwich ELISA.

Results:

FIG. 18 graphically illustrates the percentage survival of the CLPtreated animals as a function of the days after the CLP procedure. Asshown in FIG. 18, the lectin pathway deficiency in the MASP-2 (−/−) micedoes not increase the mortality of mice after polymicrobial infectionusing the cecal ligation and puncture model as compared to WT (+/+)mice. However, as shown in FIG. 19, MASP-2 (−/−) mice showed asignificantly higher bacterial load (approximately a 1000-fold increasein bacterial numbers) in peritoneal lavage after CLP when compared totheir WT (+/+) littermates. These results indicate that MASP-2 (−/−)deficient mice are resistant to septic shock. The reduced bacterialclearance in MASP-2 deficient mice in this model may be due to animpaired C3b mediated phagocytosis, as it was demonstrated that C3deposition is MASP-2 dependent.

It was determined that the TNF-alpha cytokine response to the bacterialinfection was not elevated in the MASP-2 (−/−) mice as compared to theWT (+/+) controls (data not shown). It was also determined that therewas a significantly higher serum concentration of TNF-alpha in WT (+/+)mice 16 hours after CLP in contrast to MASP-2 (−/−) mice, where theserum level of TNF-alpha remained nearly unaltered. These resultssuggest that the intense inflammatory response to the septic conditionwas tempered in MASP-2 (−/−) mice and allowed the animals to survive inthe presence of higher bacterial counts.

Taken together, these results demonstrate the potential deleteriouseffects of lectin pathway complement activation in the case ofsepticemia and the increased mortality in patients with overwhelmingsepsis. These results further demonstrate that MASP-2 deficiencymodulates the inflammatory immune response and reduces the expressionlevels of inflammatory mediators during sepsis. Therefore, it isbelieved that inhibition of MASP-2 (−/−) by administration of inhibitorymonoclonal antibodies against MASP-2 would be effective to reduce theinflammatory response in a subject suffering from septic shock.

Example 18

This Example describes analysis of MASP-2 (−/−) mice in a MurineIntranasal Infectivity Model.

Background/Rationale:

Pseudomonas aeruginosa is a Gram negative opportunistic human bacterialpathogen that causes a wide range of infections, particularly inimmune-compromised individuals. It is a major source of acquirednosocomial infections, in particular hospital-acquired pneumonia. It isalso responsible for significant morbidity and mortality in cysticfibrosis (CF) patients. P. aeruginosa pulmonary infection ischaracterized by strong neutrophil recruitment and significant lunginflammation resulting in extensive tissue damage (Palanki M. S. et al.,J. Med. Chem 51:1546-1559 (2008)).

In this Example, a study was undertaken to determine whether the removalof the lectin pathway in MASP-2 (−/−) mice increases the susceptibilityof the mice to bacterial infections.

Methods:

Twenty-two WT (+/+) mice, twenty-two MASP-2 (−/−) mice, and eleven C3(−/−) mice were challenged with intranasal administration of P.aeruginosa bacterial strain. The mice were monitored over the six dayspost-infection and Kaplan-Mayer plots were constructed showing percentsurvival.

Results:

FIG. 20 is a Kaplan-Mayer plot of the percent survival of WT (+/+),MASP-2 (−/−) or C3 (−/−) mice six days post-infection. As shown in FIG.20, no differences were observed in the MASP-2 (−/−) mice versus the WT(+/+) mice. However, removal of the classical (C1q) pathway in the C3(−/−) mice resulted in a severe susceptibility to bacterial infection.These results demonstrate that MASP-2 inhibition does not increasesusceptibility to bacterial infection, indicating that it is possible toreduce undesirable inflammatory complications in trauma patients byinhibiting MASP-2 without compromising the patient's ability to fightinfections using the classical complement pathway.

Example 19

This Example describes the pharmacodynamic analysis of representativehigh affinity anti-MASP-2 Fab2 antibodies that were identified asdescribed in Example 10.

Background/Rationale:

As described in Example 10, in order to identify high-affinityantibodies that block the rat lectin pathway, rat MASP-2 protein wasutilized to pan a phage display library. This library was designed toprovide for high immunological diversity and was constructed usingentirely human immunoglobin gene sequences. As described in Example 10,approximately 250 individual phage clones were identified that boundwith high affinity to the rat MASP-2 protein by ELISA screening.Sequencing of these clones identified 50 unique MASP-2 antibody encodingphage. Fab2 protein was expressed from these clones, purified andanalyzed for MASP-2 binding affinity and lectin complement pathwayfunctional inhibition.

As shown in TABLE 6 of Example 10, 17 anti-MASP-2 Fab2s with functionalblocking activity were identified as a result of this analysis (a 34%hit rate for blocking antibodies). Functional inhibition of the lectincomplement pathway by Fab2s was apparent at the level of C4 deposition,which is a direct measure of C4 cleavage by MASP-2. Importantly,inhibition was equally evident when C3 convertase activity was assessed,demonstrating functional blockade of the lectin complement pathway. The17 MASP-2 blocking Fab2s identified as described in Example 10 potentlyinhibit C3 convertase formation with IC₅₀ values equal to or less than10 nM. Eight of the 17 Fab2s identified have IC₅₀ values in thesub-nanomolar range. Furthermore, all 17 of the MASP-2 blocking Fab2sgave essentially complete inhibition of the C3 convertase formation inthe lectin pathway C3 convertase assay, as shown in FIGS. 8A-C, andsummarized in TABLE 6 of Example 10. Moreover, each of the 17 blockinganti-MASP-2 Fab2s shown in TABLE 6 potently inhibit C3b generation(>95%), thus demonstrating the specificity of this assay for lectinpathway C3 convertase.

Rat IgG2c and mouse IgG2a full-length antibody isotype variants werederived from Fab2 #11. This Example describes the in vivocharacterization of these isotypes for pharmacodynamic parameters.

Methods:

As described in Example 10, rat MASP-2 protein was utilized to pan a Fabphage display library, from which Fab2#11 was identified. Rat IgG2c andmouse IgG2a full-length antibody isotype variants were derived from Fab2#11. Both rat IgG2c and mouse IgG2a full length antibody isotypes werecharacterized in vivo for pharmacodynamic parameters as follows.

In Vivo Study in Mice:

A pharmacodynamic study was carried out in mice to investigate theeffect of anti-MASP-2 antibody dosing on the plasma lectin pathwayactivity in vivo. In this study, C4 deposition was measured ex vivo in alectin pathway assay at various time points following subcutaneous (sc)and intraperitoneal (ip) administration of 0.3 mg/kg or 1.0 mg/kg of themouse anti-MASP-2 MoAb (mouse IgG2a full-length antibody isotype derivedfrom Fab2#11).

FIG. 21 graphically illustrates lectin pathway specific C4b deposition,measured ex vivo in undiluted serum samples taken from mice (n=3mice/group) at various time points after subcutaneous dosing of either0.3 mg/kg or 1.0 mg/kg of the mouse anti-MASP-2 MoAb. Serum samples frommice collected prior to antibody dosing served as negative controls(100% activity), while serum supplemented in vitro with 100 nM of thesame blocking anti-MASP-2 antibody was used as a positive control (0%activity).

The results shown in FIG. 21 demonstrate a rapid and complete inhibitionof C4b deposition following subcutaneous administration of 1.0 mg/kgdose of mouse anti-MASP-2 MoAb. A partial inhibition of C4b depositionwas seen following subcutaneous administration of 0.3 mg/kg dose ofmouse anti-MASP-2 MoAb.

The time course of lectin pathway recovery was followed for three weeksfollowing a single ip administration of mouse anti-MASP-2 MoAb at 0.6mg/kg in mice. As shown in FIG. 22, a precipitous drop in lectin pathwayactivity occurred post antibody dosing followed by complete lectinpathway inhibition that lasted for about 7 days after ip administration.Slow restoration of lectin

These results demonstrate that the mouse anti-MASP-2 Moab derived fromFab2 #11 inhibits the lectin pathway of mice in a dose-responsive mannerwhen delivered systemically.

Example 20

This Example describes analysis of the mouse anti-MASP-2 Moab derivedfrom Fab2 #11 for efficacy in a mouse model for age-related maculardegeneration.

Background/Rationale:

As described in Example 10, rat MASP-2 protein was utilized to pan a Fabphage display library, from which Fab2#11 was identified as afunctionally active antibody. Full length antibodies of the rat IgG2cand mouse IgG2a isotypes were generated from Fab2 #11. The full lengthanti-MASP-2 antibody of the mouse IgG2a isotype was characterized forpharmacodynamic parameters as described in Example 19. In this Example,the mouse anti-MASP-2 full-length antibody derived from Fab2 #11 wasanalyzed in the mouse model of age-related macular degeneration (AMD),described by Bora P. S. et al, J Immunol 174:491-497 (2005).

Methods:

The mouse IgG2a full-length anti-MASP-2 antibody isotype derived fromFab2 #11 as described in Example 19, was tested in the mouse model ofage-related macular degeneration (AMD) as described in Example 13 withthe following modifications.

Administration of Mouse-Anti-MASP-2 MoAbs

Two different doses (0.3 mg/kg and 1.0 mg/kg) of mouse anti-MASP-2 MoAbalong with an isotype control MoAb treatment were injected ip into WT(+/+) mice (n=8 mice per group) 16 hours prior to CNV induction

Induction of Choroidal Neovascularization (CNV)

The induction of choroidal neovascularization (CNV) and measurement ofthe volume of CNV was carried out using laser photocoagulation asdescribed in Example 13.

Results:

FIG. 23 graphically illustrates the CNV area measured at 7 days postlaser injury in mice treated with either isotype control MoAb, or mouseanti-MASP-2 MoAb (0.3 mg/kg and 1.0 mg/kg). As shown in FIG. 23, in themice pre-treated with 1.0 mg/kg anti-MASP-2 MoAb, a statisticallysignificant (p<0.01) approximately 50% reduction in CNV was observedseven days post-laser treatment. As further shown in FIG. 23, it wasobserved that a 0.3 mg/kg dose of anti-MASP-2 MoAb was not efficaciousin reducing CNV. It is noted that the 0.3 mg/kg dose of anti-MASP-2 MoAbwas shown to have a partial and transient inhibition of C4b depositionfollowing subcutaneous administration, as described in Example 19 andshown in FIG. 21.

The results described in this Example demonstrate that blockade ofMASP-2 with an inhibitor, such as anti-MASP-2 MoAb, has a preventativeand/or therapeutic effect in the treatment of macular degeneration. Itis noted that these results are consistent with the results observed inthe study carried out in the MASP-2 (−/−) mice, described in Example 13,in which a 30% reduction in the CNV 7 days post-laser treatment wasobserved in MASP-2 (−/−) mice in comparison to the wild-type controlmice. Moreover, the results in this Example further demonstrate thatsystemically delivered anti-MASP-2 antibody provides local therapeuticbenefit in the eye, thereby highlighting the potential for a systemicroute of administration to treat AMD patients. In summary, these resultsprovide evidence supporting the use of MASP-2 MoAb in the treatment ofAMD.

Example 21

This Example demonstrates that MASP-2 deficient mice are protected fromNeisseria meningitidis induced mortality after infection with N.meningitidis and have enhanced clearance of bacteraemia as compared towild type control mice.

Rationale:

Neisseria meningitidis is a heterotrophic gram-negative diplococcalbacterium known for its role in meningitis and other forms ofmeningococcal disease such as meningococcemia. N. meningitidis is amajor cause of morbidity and mortality during childhood. Severecomplications include septicaemia, Waterhouse-Friderichsen syndrome,adrenal insufficiency and disseminated intravascular coagulation (DIC).See e.g., Rintala E. et al., Critical Care Medicine 28(7):2373-2378(2000). In this Example, the role of the lectin pathway was analyzed inMASP-2 (−/−) and WT (+/+) mice in order to address whether MASP-2deficient mice would be susceptible to N. meningitidis inducedmortality.

Methods:

MASP-2 knockout mice were generated as described in Example 1 andbackcrossed for at least 10 generations with C57Bl/6. 10 week old MASP-2KO mice (n=10) and wild type C57/B6 mice (n=10) were innoculated byintravenous injection with either a dosage of 5×10⁸ cfu/100 μl, 2×10⁸cfu/100 μl or 3×10⁷ cfu/100 μl of Neisseria meningitidis Serogroup AZ2491 in 400 mg/kg iron dextran. Survival of the mice after infectionwas monitored over a 72 hour time period. Blood samples were taken fromthe mice at hourly intervals after infection and analyzed to determinethe serum level (log cfu/ml) of N. meningitidis in order to verifyinfection and determine the rate of clearance of the bacteria from theserum.

Results:

FIG. 24A graphically illustrates the percent survival of MASP-2 KO andWT mice after administration of an infective dose of 5×10⁸/100 μl cfu N.meningitidis. As shown in FIG. 24A, after infection with the highestdose of 5×10⁸/100 μl cfu N. meningitidis, 100% of the MASP-2 KO micesurvived throughout the 72 hour period after infection. In contrast,only 20% of the WT mice were still alive 24 hours after infection. Theseresults demonstrate that MASP-2 deficient mice are protected from N.meningitidis induced mortality.

FIG. 24B graphically illustrates the log cfu/ml of N. meningitidisrecovered at different time points in blood samples taken from theMASP-2 KO and WT mice infected with 5×10⁸ cfu/100 μl N. meningitidis. Asshown in FIG. 24B, in WT mice the level of N. meningitidis in the bloodreached a peak of about 6.5 log cfu/ml at 24 hours after infection anddropped to zero by 48 hours after infection. In contrast, in the MASP-2KO mice, the level of N. meningitidis reached a peak of about 3.5 logcfu/ml at 6 hours after infection and dropped to zero by 36 hours afterinfection.

FIG. 25A graphically illustrates the percent survival of MASP-2 KO andWT mice after infection with 2×10⁸ cfu/100 μl N. meningitidis. As shownin FIG. 25A, after infection with the dose of 2×10⁸ cfu/100 μl N.meningitidis, 100% of the MASP-2 KO mice survived throughout the 72 hourperiod after infection. In contrast, only 80% of the WT mice were stillalive 24 hours after infection. Consistent with the results shown inFIG. 24A, these results further demonstrate that MASP-2 deficient miceare protected from N. meningitidis induced mortality.

FIG. 25B graphically illustrates the log cfu/ml of N. meningitidisrecovered at different time points in blood samples taken from the WTmice infected with 2×10⁸ cfu/100 μl N. meningitidis. As shown in FIG.25B, the level of N. meningitidis in the blood of WT mice infected with2×10⁸ cfu reached a peak of about 4 log cfu/ml at 12 hours afterinfection and dropped to zero by 24 hours after infection. FIG. 25Cgraphically illustrates the log cfu/ml of N. meningitidis recovered atdifferent time points in blood samples taken from the MASP-2 KO miceinfected with 2×10⁸ cfu/100 μl N. meningitidis. As shown in FIG. 25C,the level of N. meningitidis in the blood of MASP-2 KO mice infectedwith 2×10⁸ cfu reached a peak level of about 3.5 log cfu/ml at 2 hoursafter infection and dropped to zero at 3 hours after infection.Consistent with the results shown in FIG. 24B, these results demonstratethat although the MASP-2 KO mice were infected with the same dose of N.meningitidis as the WT mice, the MASP-2 KO mice have enhanced clearanceof bacteraemia as compared to WT.

The percent survival of MASP-2 KO and WT mice after infection with thelowest dose of 3×10⁷ cfu/100 μl N. meningitidis was 100% at the 72 hourtime period (data not shown).

Discussion

These results show that MASP-2 deficient mice are protected from N.meningitidis induced mortality and have enhanced clearance ofbacteraemia as compared to the WT mice. Therefore, in view of theseresults, it is expected that therapeutic application of MASP-2inhibitors, such as MASP-2 MoAb, would be expected to be efficacious totreat, prevent or mitigate the effects of infection with N. meningitidisbacteria (i.e., sepsis and DIC). Further, these results indicate thattherapeutic application of MASP-2 inhibitors, such as MASP-2 MoAb wouldnot predispose a subject to an increased risk to contract N.meningitidis infections.

Example 22

This Example describes the discovery of novel lectin pathway mediatedand MASP-2 dependent C4-bypass activation of complement C3.

Rationale:

The principal therapeutic benefit of utilizing inhibitors of complementactivation to limit myocardial ischemia/reperfusion injury (MIRI) wasconvincingly demonstrated in an experimental rat model of myocardialinfarction two decades ago: Recombinant sCRI, a soluble truncatedderivative of the cell surface complement receptor type-1 (CR1), wasgiven intravenously and its effect assessed in a rat in vivo model ofMIRI. Treatment with sCRI reduced infarct volume by more than 40%(Weisman, H. F., et al., Science 249:146-151 (1990)). The therapeuticpotential of this recombinant inhibitor was subsequently demonstrated ina clinical trial showing that the administration of sCRI in patientswith MI prevented contractile failure in the post-ischemic heart(Shandelya, S., et al., Circulation 87:536-546 (1993)). The primarymechanism leading to the activation of complement in ischemic tissue,however, has not been ultimately defined, mainly due to the lack ofappropriate experimental models, the limited understanding of themolecular processes that lead to complement activation ofoxygen-deprived cells, and the cross-talk and synergisms between thedifferent complement activation pathways.

As a fundamental component of the immune response, the complement systemprovides protection against invading microorganisms through bothantibody-dependent and -independent mechanisms. It orchestrates manycellular and humoral interactions within the immune response, includingchemotaxis, phagocytosis, cell adhesion, and B-cell differentiation.Three different pathways initiate the complement cascade: the classicalpathway, the alternative pathway, and the lectin pathway. The classicalpathway recognition subcomponent C1q binds to a variety of targets—mostprominently immune complexes—to initiate the step-wise activation ofassociated serine proteases, C1r and C1s, providing a major mechanismfor pathogen and immune complex clearance following engagement by theadaptive immune system. Binding of C1q to immune complexes converts theC1r zymogen dimer into its active form to cleave and thereby activateC1s. C1s translates C1q binding into complement activation in twocleavage steps: It first converts C4 into C4a and C4b and then cleavesC4b-bound C2 to form the C3 convertase C4b2a. This complex converts theabundant plasma component C3 into C3a and C3b. Accumulation of C3b inclose proximity of the C4b2a complex shifts the substrate specificityfor C3 to C5 to form the C5 convertase C4b2a(C3b)_(n). The C3 and C5convertase complexes generated via classical pathway activation areidentical to those generated through the lectin pathway activationroute. In the alternative pathway, spontaneous low-level hydrolysis ofcomponent C3 results in deposition of protein fragments onto cellsurfaces, triggering complement activation on foreign cells, whilecell-associated regulatory proteins on host tissues avert activation,thus preventing self-damage. Like the alternative pathway, the lectinpathway may be activated in the absence of immune complexes. Activationis initiated by the binding of a multi-molecular lectin pathwayactivation complex to Pathogen-Associated Molecular Patterns (PAMPs),mainly carbohydrate structures present on bacterial, fungal or viralpathogens or aberrant glycosylation patterns on apoptotic, necrotic,malignant or oxygen-deprived cells (Collard, C. D., et al., Am. J.Pathol. 156:1549-1556 (2000); Walport, M. J., N. Engl. J. Med.344:1058-1066 (2001); Schwaeble, W., et al., Immunobiology 205:455-466(2002); and Fujita, T., Nat. Rev. Immunol. 2:346-353 (2002)).

Mannan-binding lectin (MBL) was the first carbohydrate recognitionsubcomponent shown to form complexes with a group of novel serineproteases, named MBL-associated Serine Proteases (MASPs) and numberedaccording to the sequence of their discovery (i.e., MASP-1, MASP-2 andMASP-3). In man, lectin pathway activation complexes can be formed withfour alternative carbohydrate recognition subcomponents with differentcarbohydrate binding specificities, i.e., MBL 2, and three differentmembers of the ficolin family, namely L-Ficolin, H-ficolin and M-ficolinand MASPs. Two forms of MBL, MBL A and MBL C, and ficolin-A form lectinactivation pathway complexes with MASPs in mouse and rat plasma. We havepreviously cloned and characterised MASP-2 and an additional truncatedMASP-2 gene product of 19 kDa, termed MAp19 or sMAP, in human, mouse andrat (Thiel, S., et al., Nature 386:506-510 (1997). Stover, C. M., etal., J. Immunol. 162:3481-3490 (1999); Takahashi, M., et al., Int.Immunol. 11:859-863 (1999); and Stover, C. M., et al., J. Immunol.163:6848-6859 (1999)). MAp19/sMAP is devoid of protease activity, butmay regulate lectin pathway activation by competing for the binding ofMASPs to carbohydrate recognition complexes (Iwaki, D. et al., J.Immunol. 177:8626-8632 (2006)).

There is evidence suggesting that of the three MASPs, only MASP-2 isrequired to translate binding of the lectin pathway recognitioncomplexes into complement activation (Thiel, S., et al. (1997);Vorup-Jensen, T., et al., J. Immunol. 165:2093-2100 (2000); Thiel, S.,et al., J. Immunol. 165:878-887 (2000); Rossi, V., et al., J. Biol.Chem. 276:40880-40887 (2001)). This conclusion is underlined by thephenotype of a most recently described mouse strain deficient in MASP-1and MASP-3. Apart from a delay in the onset of lectin pathway mediatedcomplement activation in vitro—MASP-1/3 deficient mice retain lectinpathway functional activity. Reconstitution of MASP-1 and MASP-3deficient serum with recombinant MASP-1 overcomes this delay in lectinpathway activation implying that MASP-1 may facilitate MASP-2 activation(Takahashi, M., et al., J. Immunol. 180:6132-6138 (2008)). A most recentstudy has shown that MASP-1 (and probably also MASP-3) are required toconvert the alternative pathway activation enzyme Factor D from itszymogen form into its enzymatically active form (Takahashi, M., et al.,J. Exp. Med. 207:29-37 (2010)). The physiological importance of thisprocess is underlined by the absence of alternative pathway functionalactivity in plasma of MASP-1/3 deficient mice.

The recently generated mouse strains with combined targeted deficienciesof the lectin pathway carbohydrate recognition subcomponents MBL A andMBL C may still initiate lectin pathway activation via the remainingmurine lectin pathway recognition subcomponent ficolin A (Takahashi, K.,et al., Microbes Infect. 4:773-784 (2002)). The absence of any residuallectin pathway functional activity in MASP-2 deficient mice delivers aconclusive model to study the role of this effector arm of innatehumoral immunity in health and disease.

The availability of C4 and MASP-2 deficient mouse strains allowed us todefine a novel lectin pathway specific, but MASP-2 dependent, C4-bypassactivation route of complement C3. The essential contribution of thisnovel lectin pathway mediated C4-bypass activation route towardspost-ischemic tissue loss is underlined by the prominent protectivephenotype of MASP-2 deficiency in MIRI while C4-deficient mice tested inthe same model show no protection.

In this Example, we describe a novel lectin pathway mediated and MASP-2dependent C4-bypass activation of complement C3. The physiologicalrelevance of this new activation route is established by the protectivephenotype of MASP-2 deficiency in an experimental model of myocardialischemia/reperfusion injury (MIRI), where C4 deficient animals were notprotected.

Methods:

MASP-2 Deficient Mice Show No Gross Abnormalities.

MASP-2 deficient mice were generated as described in Example 1. Bothheterozygous (^(+/−)) and homozygous (^(−/−)) MASP-2 deficient mice arehealthy and fertile, and show no gross abnormalities. Their lifeexpectancy is similar to that of their WT littermates (>18 months).Prior to studying the phenotype of these mice in experimental models ofdisease, our MASP-2^(−/−) line was backcrossed for eleven generationsonto a C57BL/6 background. The total absence of MASP-2 mRNA wasconfirmed by Northern blotting of poly A+ selected liver RNApreparations, while the 1.2 kb mRNA encoding MAp19 or sMAP (a truncatedalternative splicing product of the MASP2 gene) is abundantly expressed.

qRT-PCR analysis using primer pairs specific for either the codingsequence for the serine protease domain of MASP-2 (B chain) or theremainder of the coding sequence for the A-chain showed that no B chainencoding mRNA is detectable in MASP-2^(−/−) mice while the abundance ofthe disrupted A chain mRNA transcript was significantly increased.Likewise, the abundance of MAp19/sMAP encoding mRNA is increased inMASP-2^(+/−) and MASP-2_(−/−) mice. Plasma MASP-2 levels, determined byELISA for 5 animals of each genotype, were 300 ng/ml for WT controls(range 260-330 ng/ml), 360 ng/ml for heterozygous mice (range 330-395ng/ml) and undetectable inMASP-2^(−/−) mice. Using qRT-PCR, mRNAexpression profiles were established demonstrating that MASP-2^(−/−)mice express mRNA for MBL A, MBL C, ficolin A, MASP-1, MASP-3, C1q,C1rA, ClsA, Factor B, Factor D, C4, and C3 at an abundance similar tothat of their MASP-2 sufficient littermates (data not shown).

Plasma C3 levels of MASP-2^(−/−) (n=8) and MASP-2^(+/+) (n=7)littermates were measured using a commercially available mouse C3 ELISAkit (Kamiya, Biomedical, Seattle, Wash.). C3 levels of MASP-2 deficientmice (average 0.84 mg/ml, +/−0.34) were similar to those of the WTcontrols (average 0.92, +/−0.37).

Results:

MASP-2 is Essential for Lectin Pathway Functional Activity.

As described in Example 2 and shown in FIG. 5, the in vitro analyses ofMASP-2^(−/−)plasma showed a total absence of lectin pathway functionalactivity on activating Mannan- and Zymosan-coated surfaces for theactivation of C4. Likewise, neither lectin pathway-dependent C4 nor C3cleavage was detectable in MASP-2_(−/−) plasma on surfaces coated withN-acetyl glucosamine, which binds and triggers activation via MBL A, MBLC and ficolin A (data not shown).

The analyses of sera and plasma of MASP-2−/−mice clearly demonstratedthat MASP-2 is essentially required to activate complement via thelectin pathway. The total deficiency of lectin pathway functionalactivity, however, leaves the other complement activation pathwaysintact: MASP-2−/−plasma can still activate complement via the classical(FIG. 26A) and the alternative pathway (FIG. 26B). In FIGS. 26A and 26B,the symbol “*” symbol indicates serum from WT (MASP-2 (+/+)); the symbol“▪” indicates serum from WT (C1q depleted); the symbol “□” indicatesserum from MASP-2 (−/−); and the symbol “Δ” indicates serum from MASP-2(−/−) (C1q depleted).

FIG. 26A graphically illustrates that MASP-2−/− mice retain a functionalclassical pathway: C3b deposition was assayed on microtiter platescoated with immune complexes (generated in situ by coating with BSA thenadding goat anti-BSA IgG). FIG. 26B graphically illustrates MASP-2deficient mice retain a functional alternative pathway: C3b depositionwas assayed on Zymosan coated microtiter plates under conditions thatpermit only alternative pathway activation (buffer containing Mg²⁺ andEGTA). Results shown in FIG. 26A and FIG. 26B are means of duplicatesand are typical of three independent experiments. Same symbols forplasma sources were used throughout. These results show that afunctional alternative pathway is present in MASP-2 deficient mice, asevidenced in the results shown in FIG. 26B under experimental conditionsdesigned to directly trigger the alternative pathway, while inactivatingboth the classical pathway and lectin pathway.

The Lectin Pathway of Complement Activation Critically Contributes toInflammatory Tissue Loss in Myocardial Ischemia/Reperfusion Injury(MIRI).

In order to study the contribution of lectin pathway functional activityto MIRI, we compared MASP-2^(−/−) mice and WT littermate controls in amodel of MIRI following transient ligation and reperfusion of the leftanterior descending branch of the coronary artery (LAD). The presence orabsence of complement C4 has no impact on the degree of ischemic tissueloss in MIRI. We assessed the impact of C4 deficiency on infarct sizesfollowing experimental MIRI. As shown in FIG. 27A and FIG. 27B,identical infarct sizes were observed in both C4-deficient mice andtheir WT littermates. FIG. 27A graphically illustrates MIRI-inducedtissue loss following LAD ligation and reperfusion in C4−/− mice (n=6)and matching WT littermate controls (n=7). FIG. 27B graphicallyillustrates INF as a function of AAR, clearly demonstrating that C4−/−mice are as susceptible to MIRI as their WT controls (dashed line).

These results demonstrate that C4 deficient mice are not protected fromMIRI. This result was unexpected, as it is in conflict with the widelyaccepted view that the major C4 activation fragment, C4b, is anessential component of the classical and the lectin pathway C3convertase C4b2a. We therefore assessed whether a residual lectinpathway specific activation of complement C3 can be detected inC4-deficient mouse and human plasma.

The Lectin Pathway can Activate Complement C3 in Absence of C4 Via aNovel MASP-2 Dependent C4-Bypass Activation Route.

Encouraged by historical reports indicating the existence of a C4-bypassactivation route in C4-deficient guinea pig serum (May, J. E., and M.Frank, J. Immunol. 111:1671-1677 (1973)), we analyzed whetherC4-deficient mice may have residual classical or lectin pathwayfunctional activity and monitored activation of C3 underpathway-specific assay conditions that exclude contributions of thealternative pathway.

C3b deposition was assayed on Mannan-coated microtiter plates usingre-calcified plasma at plasma concentrations prohibitive for alternativepathway activation (1.25% and below). While no cleavage of C3 wasdetectable in C4-deficient plasma tested for classical pathwayactivation (data not shown), a strong residual C3 cleavage activity wasobserved in C4-deficient mouse plasma when initiating complementactivation via the lectin pathway. The lectin pathway dependence isdemonstrated by competitive inhibition of C3 cleavage followingpreincubation of C4-deficient plasma dilutions with soluble Mannan (seeFIG. 28A). As shown in FIG. 28A-D, MASP-2 dependent activation of C3 wasobserved in the absence of C4. FIG. 28A graphically illustrates C3bdeposition by C4+/+ (crosses) and C4−/− (open circles) mouse plasma.Pre-incubating the C4−/− plasma with excess (1 μg/ml) fluid-phase Mannanprior to the assay completely inhibits C3 deposition (filled circles).Results are typical of 3 independent experiments. FIG. 28B graphicallyillustrates the results of an experiment in which wild-type, MASP-2deficient (open squares) and C4−/−mouse plasma (1%) was mixed withvarious concentrations of anti-rat MASP-2 mAbM11 (abscissa) and C3bdeposition assayed on Mannan-coated plates. Results are means (±SD) of 4assays (duplicates of 2 of each type of plasma). FIG. 28C graphicallyillustrates the results of an experiment in which Human plasma: pooledNHS (crosses), C4−/− plasma (open circles) and C4−/− plasmapre-incubated with 1 μg/ml Mannan (filled circles). Results arerepresentative of three independent experiments. FIG. 28D graphicallyillustrates that inhibition of C3b deposition in C4 sufficient and C4deficient human plasma (1%) by anti-human MASP-2 mAbH3 (Means±SD oftriplicates). As shown in FIG. 28B, no lectin pathway-dependent C3activation was detected in MASP-2−/− plasma assayed in parallel,implying that this C4-bypass activation route of C3 is MASP-2 dependent.

To further corroborate these findings, we established a series ofrecombinant inhibitory mAbs isolated from phage display antibodylibraries by affinity screening against recombinant human and ratMASP-2A (where the serine residue of the active protease domain wasreplaced by an alanine residue by site-directed mutagenesis to preventautolytic degradation of the antigen). Recombinant antibodies againstMASP-2 (AbH3 and AbMll) were isolated from Combinatorial AntibodyLibraries (Knappik, A., et al., J. Mol. Biol. 296:57-86 (2000)), usingrecombinant human and rat MASP-2A as antigens (Chen, C. B. and Wallis,J. Biol. Chem. 276:25894-25902 (2001)). An anti-rat Fab2 fragment thatpotently inhibited lectin pathway-mediated activation of C4 and C3 inmouse plasma (IC50˜1 nM) was converted to a full-length IgG2a antibody.Polyclonal anti-murine MASP-2A antiserum was raised in rats. These toolsallowed us to confirm MASP-2 dependency of this novel lectin pathwayspecific C4-bypass activation route of C3, as further described below.

As shown in FIG. 28B, M211, an inhibitory monoclonal antibody whichselectively binds to mouse and rat MASP-2 inhibited the C4-bypassactivation of C3 in C4-deficient mouse as well as C3 activation of WTmouse plasma via the lectin pathway in a concentration dependent fashionwith similar IC₅ values. All assays were carried out at high plasmadilutions rendering the alternative pathway activation routedysfunctional (with the highest plasma concentration being 1.25%).

In order to investigate the presence of an analogous lectin pathwayspecific C4-bypass activation of C3 in humans, we analyzed the plasma ofa donor with an inherited deficiency of both human C4 genes (i.e., C4Aand C4B), resulting in total absence of C4 (Yang, Y., et al., J.Immunol. 173:2803-2814 (2004)). FIG. 28C shows that this patient'splasma efficiently activates C3 in high plasma dilutions (rendering thealternative activation pathway dysfunctional). The lectin pathwayspecific mode of C3 activation on Mannan-coated plates is demonstratedin murine C4-deficient plasma (FIG. 28A) and human C4 deficient plasma(FIG. 28C) by adding excess concentrations of fluid-phase Mannan. TheMASP-2 dependence of this activation mechanism of C3 in humanC4-deficient plasma was assessed using AbH3, a monoclonal antibody thatspecifically binds to human MASP-2 and ablates MASP-2 functionalactivity. As shown in FIG. 28D, AbH3 inhibited the deposition of C3b(and C3dg) in both C4-sufficient and C4-deficient human plasma withcomparable potency.

In order to assess a possible role of other complement components in theC4-bypass activation of C3, we tested plasma of MASP-1/3−/− and Bf/C2−/−mice alongside MASP-2−/−, C4−/− and C1q−/− plasma (as controls) underboth lectin pathway specific and classical pathway specific assayconditions. The relative amount of C3 cleavage was plotted against theamount of C3 deposited when using WT plasma.

FIG. 29A graphically illustrates a comparative analysis of C3 convertaseactivity in plasma from various complement deficient mouse strainstested either under lectin activation pathway or classical activationpathway specific assay conditions. Diluted plasma samples (1%) of WTmice (n=6), MASP-2−/−mice (n=4), MASP-1/3−/− mice (n=2), C4−/− mice(n=8), C4/MASP-1/3−/− mice (n=8), Bf/C2−/− (n=2) and C1q−/− mice (n=2)were tested in parallel. Reconstitution of Bf/C2−/− plasma with 2.5μg/ml recombinant rat C2 (Bf/C2−/−+C2) restored C3b deposition. Resultsare means (+SD). **p<0.01 (compared to WT plasma). As shown in FIG. 29A,substantial C3 deposition is seen in C4−/− plasma tested under lectinpathway specific assay conditions, but not under classical pathwayspecific conditions. Again, no C3 deposition was seen in MASP-2deficient plasma via the lectin pathway activation route, while the sameplasma deposited C3 via the classical pathway. In MASP-1/3−/− plasma, C3deposition occurred in both lectin and classical pathway specific assayconditions. No C3 deposition was seen in plasma with a combineddeficiency of C4 and MASP-1/3, either using lectin pathway or classicalpathway specific conditions. No C3 deposition is detectable in C2/Bf−/−plasma, either via the lectin pathway, or via the classical pathway.Reconstitution of C2/Bf−/− mouse plasma with recombinant C2, however,restored both lectin pathway and classical pathway-mediated C3 cleavage.The assay conditions were validated using C1q−/− plasma.

FIG. 29B graphically illustrates time-resolved kinetics of C3 convertaseactivity in plasma from various complement deficient mouse strains WT,fB−/−, C4−/−, MASP-1/3−/−, and MASP-2−/−plasma, tested under lectinactivation pathway specific assay conditions (1% plasma, results aretypical of three independent experiments). As shown in FIG. 29B, whileno C3 cleavage was seen in MASP-2−/−plasma, fB−/− plasma cleaved C3 withsimilar kinetics to the WT plasma. A significant delay in the lectinpathway-dependent conversion of C3 to C3b (and C3dg) was seen in C4−/−as well as in MASP-1/3 deficient plasma. This delay of C3 activation inMASP-1/3−/− plasma was recently shown to be MASP-1, rather than MASP-3dependent (Takahashi, M., et al., J. Immunol. 180:6132-6138 (2008)).

Discussion

The results described in this Example strongly suggest that MASP-2functional activity is essential for the activation of C3 via the lectinpathway both in presence and absence of C4. Furthermore, C2 and MASP-1are required for this novel lectin pathway specific C4-bypass activationroute of C3 to work. The comparative analysis of lectin pathwayfunctional activity in MASP-2−/− as well as C4−/− plasma revealed theexistence of a previously unrecognized C4-independent, butMASP-2-dependent activation route of complement C3 and showed that C3can be activated in a lectin pathway-dependent mode in total absence ofC4. While the detailed molecular composition and the sequence ofactivation events of this novel MASP-2 dependent C3 convertase remainsto be elucidated, our results imply that this C4-bypass activation routeadditionally requires the presence of complement C2 as well as MASP-1.The loss of lectin pathway-mediated C3 cleavage activity in plasma ofmice with combined C4 and MASP-1/3 deficiency may be explained by a mostrecently described role of MASP-1 to enhance MASP-2 dependent complementactivation through direct cleavage and activation of MASP-2 (Takahashi,M., et al., J. Immunol. 180:6132-6138 (2008)). Likewise, MASP-1 may aidMASP-2 functional activity through its ability to cleave C2(Moller-Kristensen, et al., Int. Immunol. 19:141-149 (2007)). Bothactivities may explain the reduced rate by which MASP-1/3 deficientplasma cleaves C3 via the lectin activation pathway and why MASP-1 maybe required to sustain C3 conversion via the C4-bypass activation route.

The inability of C2/fB−/− plasma to activate C3 via the lectin pathwaywas shown to be C2-dependent as the addition of recombinant rat C2 toC2/fB−/− plasma restored the ability of the reconstituted plasma toactivate C3 on Mannan-coated plates.

The finding that C4 deficiency specifically disrupts the classicalcomplement activation pathway while the lectin pathway retains aphysiologically critical level of C3 convertase activity via a MASP-2dependent C4-bypass activation route calls for a re-assessment of therole of the lectin pathway in various disease models, includingexperimental S. pneumoniae infection (Brown, J. S., et al., Proc. Natl.Acad. Sci. U.S.A 99:16969-16974 (2002); Experimental AllergicEncephalomyelitis (Boos, L. A., et al., Glia 49:158-160 (2005); andmodels of C3 dependent murine liver regeneration (Clark, A., et al.,Mol. Immunol. 45:3125-3132 (2008)). The latter group demonstrated thatC4-deficient mice can activate C3 in an alternative pathway independentfashion as in vivo inhibition of the alternative pathway by anantibody-mediated depletion of factor B functional activity did noteffect C3 cleavage-dependent liver regeneration in C4−/− mice (Clark,A., et al. (2008)). This lectin pathway mediated C4-bypass activationroute of C3 may also explain the lack of a protective phenotype of C4deficiency in our model of MIRI as well as in a previously describedmodel of renal allograft rejection (Lin, T., et al., Am. J. Pathol.168:1241-1248 (2006)). In contrast, our recent results haveindependently demonstrated a significant protective phenotype ofMASP-2−/−mice in models of renal transplantation (Farrar, C. A., et al.,Mol. Immunol. 46:2832 (2009)).

In summary, the results of this Example support the view that MASP-2dependent C4-bypass activation of C3 is a physiologically relevantmechanism that may be important under conditions where availability ofC4 is limiting C3 activation.

Example 23

This Example describes activation of C3 by thrombin substrates and C3deposition on mannan in WT (+/+), MASP-2 (−/−), F11 (−/−), F11/C4 (−/−)and C4 (−/−) mice.

Rationale:

As described in Example 14, it was determined that thrombin activationcan occur following lectin pathway activation under physiologicalconditions, and demonstrates the extent of MASP-2 involvement. C3 playsa central role in the activation of complement system. C3 activation isrequired for both classical and alternative complement activationpathways. An experiment was carried out to determine whether C3 isactivated by thrombin substrates.

Methods:

C3 Activation by Thrombin Substrates

Activation of C3 was measured in the presence of the following activatedforms of thrombin substrates; human FCXIa, human FVIIa, bovine FXa,human FXa, human activated protein C, and human thrombin. C3 wasincubated with the various thrombin substrates, then separated underreducing conditions on 10% SDS-polyacrylamide gels. Afterelectrophoretic transfer using cellulose membrane, the membrane wasincubated with monoclonal biotin-coupled rat anti-mouse C3, detectedwith a streptavidin-HRP kit and developed using ECL reagent.

Results:

Activation of C3 involves cleavage of the intact a-chain into thetruncated a′ chain and soluble C3a (not shown in FIG. 30). FIG. 30 showsthe results of a Western blot analysis on the activation of human C3 bythrombin substrates, wherein the uncleaved C3 alpha chain, and theactivation product a′ chain are shown by arrows. As shown in FIG. 30,incubation of C3 with the activated forms of human clotting factor XIand factor X, as well as activated bovine clotting factor X, can cleaveC3 in vitro in the absence of any complement proteases.

C3 Deposition on Mannan

C3 deposition assays were carried out on serum samples obtained from WT,MASP-2 (−/−), F11(−/−), F11(−/−)/C4(−/−) and C4(−/−). F11 is the geneencoding coagulation factor XI. To measure C3 activation, microtiterplates were coated with mannan (1 μg/well), then adding sheep anti-HSAserum (2 pig/ml) in TBS/tween/Ca²⁺. Plates were blocked with 0.1% HSA inTBS and washed as above. Plasma samples were diluted in 4 mM barbital,145 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4, added to the plates andincubated for 1.5 h at 37° C. After washing, bound C3b was detectedusing rabbit anti-human C3c (Dako), followed by alkalinephosphatase-conjugated goat anti-rabbit IgG and pNPP.

Results:

FIG. 31 shows the results of the C3 deposition assay on serum samplesobtained 5 from WT, MASP-2 (−/−), F11(−/−), F11(−/−)/C4 (−/−) and C4(−/−). As shown in FIG. 31, there is a functional lectin pathway even inthe complete absence of C4. As further shown in FIG. 31, this novellectin pathway dependent complement activation requires coagulationfactor XI.

Discussion

Prior to the results obtained in this experiment, it was believed bythose in the art that the lectin pathway of complement required C4 foractivity. Hence, data from C4 knockout mice (and C4 deficient humans)were interpreted with the assumption that such organisms were lectinpathway deficient (in addition to classical pathway deficiency). Thepresent results demonstrate that this notion is false. Thus, conclusionsof past studies suggesting that the lectin pathway was not important incertain disease settings based on the phenotype of C4 deficient animalsmay be false. The data described in this Example also show that in thephysiological context of whole serum the lectin pathway can activatecomponents of the coagulation cascade. Thus, it is demonstrated thatthere is cross-talk between complement and coagulation involving MASP-2.

Example 24

This Example describes methods to assess the effect of an anti-MASP-2antibody on lysis of red blood cells from blood samples obtained fromParoxysmal nocturnal hemoglobinuria (PNH) patients.

Background/Rationale:

Paroxysmal nocturnal hemoglobinuria (PNH), also referred to asMarchiafava-Micheli syndrome, is an acquired, potentiallylife-threatening disease of the blood, characterized bycomplement-induced intravascular hemolytic anemia. The hallmark of PNHis chronic intravascular hemolysis that is a consequence of unregulatedactivation of the alternative pathway of complement. Lindorfer, M. A.,et al., Blood 115(11) (2010). Anemia in PNH is due to destruction of redblood cells in the bloodstream. Symptoms of PNH include red urine, dueto appearance of hemoglobin in the urine, and thrombosis. PNH maydevelop on its own, referred to as “primary PNH” or in the context ofother bone marrow disorders such as aplastic anemia, referred to as“secondary PNH”. Treatment for PNH includes blood transfusion foranemia, anticoagulation for thrombosis and the use of the monoclonalantibody eculizumab (Soliris), which protects blood cells against immunedestruction by inhibiting the complement system (Hillmen P. et al., N.Engl. J. Med. 350(6):552-9 (2004)). However, a significant portion ofPNH patients treated with eculizumab are left with clinicallysignificant immune-mediated hemolytic anemia because the antibody doesnot block activation of the alternative pathway of complement.

This Example describes methods to assess the effect of an anti-MASP-2antibody on lysis of red blood cells from blood samples obtained fromPNH patients (not treated with Soliris) that are incubated withABO-matched acidified normal human serum.

Methods:

Reagents:

Erythrocytes from normal donors and from patients suffering from PNH(not treated with Soliris) are obtained by venipuncture, and prepared asdescribed in Wilcox, L. A., et al., Blood 78:820-829 (1991), herebyincorporated herein by reference. Anti-MASP-2 antibodies with functionalblocking activity of the lectin pathway may be generated as described inExample 10.

Hemolysis Analysis:

The method for determining the effect of anti-MASP-2 antibodies on theability to block hemolysis of erythrocytes from PNH patients is carriedout using the methods described in Lindorfer, M. A., et al., Blood15(11):2283-91 (2010) and Wilcox, L. A., et al., Blood 78:820-829(1991), both references hereby incorporated herein by reference. Asdescribed in Lindorfer et al., erythrocytes from PNH patient samples arecentrifuged, the buffy coat is aspirated and the cells are washed ingelatin veronal buffer (GVB) before each experiment. The erythrocytesare tested for susceptibility to APC-mediated lysis as follows.ABO-matched normal human sera are diluted with GVB containing 0.15 mMCaCl₂ and 0.5 mM MgCl₂ (GVB⁺²) and acidified to pH 6.4 (acidified NHS,aNHS) and used to reconstitute the erythrocytes to a hematocrit of 1.6%in 50% aNHS. The mixtures are then incubated at 37° C., and after 1hour, the erythrocytes are pelleted by centrifugation. The opticaldensity of an aliquot of the recovered supernate is measured at 405 nMand used to calculate the percent lysis. Samples reconstituted inacidified serum-EDTA are processed similarly and used to definebackground noncomplement-mediated lysis (typically less than 3%).Complete lysis (100%) is determined after incubating the erythrocytes indistilled water.

In order to determine the effect of anti-MASP-2 antibodies on hemolysisof PNH erythrocytes, erythrocytes from PNH patients are incubated inaNHS in the presence of incremental concentrations of the anti-MASP-2antibodies, and the presence/amount of hemolysis is subsequentlyquantified.

In view of the fact that anti-MASP-2 antibodies have been shown to blocksubsequent activation of the alternative complement pathway, it isexpected that anti-MASP-2 antibodies will be effective in blockingalternative pathway-mediated hemolysis of PNH erythrocytes, and will beuseful as a therapeutic to treat patients suffering from PNH.

Example 25

This Example describes methods to assess the effect of an anti-MASP-2blocking antibody on complement activation by cryoglobulins in bloodsamples obtained from patients suffering from cryoglobulinemia.

Background/Rationale:

Cryoglobulinemia is characterized by the presence of cryoglobulins inthe serum. Cryoglobulins are single or mixed immunoglobulins (typicallyIgM antibodies) that undergo reversible aggregation at low temperatures.Aggregation leads to classical pathway complement activation andinflammation in vascular beds, particularly in the periphery. Clinicalpresentations of cryoglobulinemia include vasculitis andglomerulonephritis.

Cryoglobulinemia may be classified as follows based on cryoglobulincomposition: Type I cryoglobulinemia, or simple cryoglobulinemia, is theresult of a monoclonal immunoglobulin, usually immunoglobulin M (IgM);Types II and III cryoglobulinemia (mixed cryoglobulinemia) containrheumatoid factors (RFs), which are usually IgM in complexes with the Fcportion of polyclonal IgG.

Conditions associated with cryoglobulinemia include hepatitis Cinfection, lymphoproliferative disorders and other autoimmune diseases.Cryoglobulin-containing immune complexes result in a clinical syndromeof systemic inflammation, possibly due to their ability to activatecomplement. While IgG immune complexes normally activate the classicalpathway of complement, IgM containing complexes can also activatecomplement via the lectin pathway (Zhang, M., et al., Mol Immunol44(1-3):103-110 (2007) and Zhang. M., et al., J. Immunol. 177(7):4727-34(2006)).

Immunohistochemical studies have further demonstrated the cryoglobulinimmune complexes contain components of the lectin pathway, and biopsiesfrom patients with cryoglobulinemic glomerulonephritis showedimmunohistochemical evidence of lectin pathway activation in situ(Ohsawa, I., et al., Clin Immunol 101(1):59-66 (2001)). These resultssuggest that the lectin pathway may contribute to inflammation andadverse outcomes in cryoglobulemic diseases.

Methods:

The method for determining the effect of anti-MASP-2 antibodies on theability to block the adverse effects of Cryoglobulinemia is carried outusing the assay for fluid phase C3 conversion as described in Ng Y. C.et al., Arthritis and Rheumatism 31(1):99-107 (1988), herebyincorporated herein by reference. As described in Ng et al., inessential mixed cryoglobulinemia (EMC), monoclonal rheumatoid factor(mRF), usually IgM, complexes with polyclonal IgG to form thecharacteristic cryoprecipitate immune complexes (IC) (type IIcryoglobulin). Immunoglobulins and C3 have been demonstrated in vesselwalls in affected tissues such as skin, nerve and kidney. As describedin Ng et al., ¹²⁵I-labeled mRF is added to serum (normal human serum andserum obtained from patients suffering from cryoglobulinemia), incubatedat 37° C., and binding to erythrocytes is measured.

Fluid phase C3 conversion is determined in serum (normal human serum andserum obtained from patients suffering from cryoglobulinemia) in thepresence or absence of the following IC: BSA-anti BSA, mRF, mRF plusIgG, or cryoglobulins, in the presence or absence of anti-MASP-2antibodies. The fixation of C3 and C4 to IC is measured using acoprecipitation assay with F(ab′)₂ anti-C3 and F(ab′)₂ anti-C4.

In view of the fact that anti-MASP-2 antibodies have been shown to blockactivation of the lectin pathway it is expected that anti-MASP-2antibodies will be effective in blocking complement mediated adverseeffects associated with cryoglobulinemia, and will be useful as atherapeutic to treat patients suffering from cryoglobulinemia.

Example 26

This Example describes methods to assess the effect of an anti-MASP-2antibody on blood samples obtained from patients with Cold AgglutininDisease, which manifests as anemia.

Background/Rationale:

Cold Agglutinin Disease (CAD), is a type of autoimmune hemolytic anemia.Cold agglutinins antibodies (usually IgM) are activated by coldtemperatures and bind to and aggregate red blood cells. The coldagglutinin antibodies combine with complement and attack the antigen onthe surface of red blood cells. This leads to opsoniation of red bloodcells (hemolysis) which triggers their clearance by thereticuloendothelial system. The temperature at which the agglutinationtakes place varies from patient to patient.

CAD manifests as anemia. When the rate of destruction of red blood celldestruction exceeds the capacity of the bone marrow to produce anadequate number of oxygen-carrying cells, then anemia occurs. CAD can becaused by an underlying disease or disorder, referred to as “SecondaryCAD”, such as an infectious disease (mycoplasma pneumonia, mumps,mononucleosis), lymphoproliferative disease (lymphoma, chroniclymphocytic leukemia), or connective tissue disorder. Primary CADpatients are considered to have a low grade lymphoproliferative bonemarrow disorder. Both primary and secondary CAD are acquired conditions.

Methods:

Reagents:

Erythrocytes from normal donors and from patients suffering from CAD areobtained by venipuncture. Anti-MASP-2 antibodies with functionalblocking activity of the lectin pathway may be generated as described inExample 10.

The effect of anti-MASP-2 antibodies to block cold aggultinin-mediatedactivation of the lectin pathway may be determined as follows.Erythrocytes from blood group I positive patients are sensitized withcold aggultinins (i.e., IgM antibodies), in the presence or absence ofanti-MASP-2 antibodies. The erythrocytes are then tested for the abilityto activate the lectin pathway by measuring C3 binding.

In view of the fact that anti-MASP-2 antibodies have been shown to blockactivation of the lectin pathway, it is expected that anti-MASP-2antibodies will be effective in blocking complement mediated adverseeffects associated with Cold Agglutinin Disease, and will be useful as atherapeutic to treat patients suffering from Cold Agglutinin Disease.

Example 27

This Example describes methods to assess the effect of an anti-MASP-2antibody on lysis of red blood cells in blood samples obtained from micewith atypical hemolytic uremic syndrome (aHUS).

Background/Rationale:

Atypical hemolytic uremic syndrome (aHUS) is characterized by hemolyticanemia, thrombocytopenia, and renal failure caused by platelet thrombiin the microcirculation of the kidney and other organs. aHUS isassociated with defective complement regulation and can be eithersporadic or familial. aHUS is associated with mutations in genes codingfor complement activation, including complement factor H, membranecofactor B and factor I, and well as complement factor H-related 1(CFHR1) and complement factor H-related 3 (CFHR3). Zipfel, P. F., etal., PloS Genetics 3(3):e41 (2007). This Example describes methods toassess the effect of an anti-MASP-2 antibody on lysis of red blood cellsfrom blood samples obtained from aHUS mice.

Methods:

The effect of anti-MASP-2 antibodies to treat aHUS may be determined ina mouse model of this disease in which the endogenouse mouse fH gene hasbeen replaced with a human homologue encoding a mutant form of fHfrequently found in aHUS patients. See Pickering M. C. et al., J. Exp.Med. 204(6):1249-1256 (2007), hereby incorporated herein by reference.As described in Pickering et al., such mice develop an aHUS likepathology. In order to assess the effect of an anti-MASP-2 antibody forthe treatment of aHUS, anti-MASP-2 antibodies are administered to themutant aHUS mice and lysis of red blood cells obtained from anti-MASP-2ab treated and untreated controls is compared. In view of the fact thatanti-MASP-2 antibodies have been shown to block activation of the lectinpathway it is expected that anti-MASP-2 antibodies will be effective inblocking lysis of red blood cells in mammalian subjects suffering fromaHUS.

Example 28

This Example describes methods to assess the effect of an anti-MASP-2antibody for the treatment of glaucoma.

Rationale/Background:

It has been shown that uncontrolled complement activation contributes tothe progression of degenerative injury to retinal ganglion cells (RGCs),their synapses and axons in glaucoma. See Tezel G. et al., InvestOphthalmol Vis Sci 51:5071-5082 (2010). For example, histopathologicstudies of human tissues and in vivo studies using different animalmodels have demonstrated that complement components, including C1q andC3, are synthesized and terminal complement complex is formed in theglaucomatous retina (see Stasi K. et al., Invest Ophthalmol Vis Sci47:1024-1029 (2006), Kuehn M. H. et al., Exp Eye Res 83:620-628 (2006)).As further described in Kuehn M. H. et al., Experimental Eye Research87:89-95 (2008), complement synthesis and deposition is induced byretinal I/R and the disruption of the complement cascade delays RGCdegeneration. In this study, mice carrying a targeted disruption of thecomplement component C3 were found to exhibit delayed RGC degenerationafter transient retinal IR when compared to normal animals.

Methods:

The method for determining the effect of anti-MASP-2 antibodies on RGCdegeneration is carried out in an animal model of retinal I/R asdescribed in Kuehn M. H. et al., Experimental Eye Research 87:89-95(2008), hereby incorporated herein by reference. As described in Kuehnet al., retinal ischemia is induced by anesthetizing the animals, theninserting a 30-gauge needle connected to a reservoir containingphosphate buffered saline through the cornea into the anterior chamberof the eye. The saline reservoir is then elevated to yield anintraocular pressure of 104 mmHg, sufficient to completely preventcirculation through the retinal vasculature. Elevated intraocularischemia is confirmed by blanching of the iris and retina and ischemiais maintained for 45 minutes in the left eye only; the right eye servesas a control and does not receive cannulation. Mice are then euthanizedeither 1 or 3 weeks after the ischemic insult. Anti-MASP-2 antibodiesare administered to the mice either locally to the eye or systemicallyto assess the effect of an anti-MASP antibody administered prior toischemic insult.

Immunohistochemistry of the eyes is carried out using antibodies againstC1q and C3 to detect complement deposition. Optic nerve damage can alsobe assessed using standard electron microscopy methods. Quantitation ofsurviving retinal RGCs is performed using gamma synuclein labeling.

Results:

As described in Kuehn et al., in normal control mice, transient retinalischemia results in degenerative changes of the optic nerve and retinaldeposits of C1q and C3 detectable by immunohistochemistry. In contrast,C3 deficient mice displayed a marked reduction in axonal degeneration,exhibiting only minor levels of optic nerve damage 1 week afterinduction. Based on these results, it is expected that similar resultswould be observed when this assay is carried out in a MASP-2 knockoutmouse, and when anti-MASP-2 antibodies are administered to a normalmouse prior to ischemic insult.

Example 29

This Example demonstrates that a MASP-2 inhibitor, such as ananti-MASP-2 antibody, is effective for the treatment of radiationexposure and/or for the treatment, amelioration or prevention of acuteradiation syndrome.

Rationale:

Exposure to high doses of ionizing radiation causes mortality by twomain mechanisms: toxicity to the bone marrow and gastrointestinalsyndrome. Bone marrow toxicity results in a drop in all hematologiccells, predisposing the organism to death by infection and hemorrhage.The gastrointestinal syndrome is more severe and is driven by a loss ofintestinal barrier function due to disintegration of the gut epitheliallayer and a loss of intestinal endocrine function. This leads to sepsisand associated systemic inflammatory response syndrome which can resultin death.

The lectin pathway of complement is an innate immune mechanism thatinitiates inflammation in response to tissue injury and exposure toforeign surfaces (i.e., bacteria). Blockade of this pathway leads tobetter outcomes in mouse models of ischemic intestinal tissue injury orseptic shock. It is hypothesized that the lectin pathway may triggerexcessive and harmful inflammation in response to radiation-inducedtissue injury. Blockade of the lectin pathway may thus reduce secondaryinjury and increase survival following acute radiation exposure.

The objective of the study carried out as described in this Example wasto assess the effect of lectin pathway blockade on survival in a mousemodel of radiation injury by administering anti-murine MASP-2antibodies.

Methods and Materials:

Materials.

The test articles used in this study were (i) a high affinityanti-murine MASP-2 antibody (mAbM11) and (ii) a high affinity anti-humanMASP-2 antibody (mAbH6) that block the MASP-2 protein component of thelectin complement pathway which were produced in transfected mammaliancells. Dosing concentrations were 1 mg/kg of anti-murine MASP-2 antibody(mAbM11), 5 mg/kg of anti-human MASP-2 antibody (mAbH6), or sterilesaline. For each dosing session, an adequate volume of fresh dosingsolutions were prepared.

Animals.

Young adult male Swiss-Webster mice were obtained from HarlanLaboratories (Houston, Tex.). Animals were housed in solid-bottom cageswith Alpha-Dri bedding and provided certified PMI 5002 Rodent Diet(Animal Specialties, Inc., Hubbard Oreg.) and water ad libitum.Temperature was monitored and the animal holding room operated with a 12hour light/12 hour dark light cycle.

Irradiation.

After a 2-week acclimation in the facility, mice were irradiated at 6.5and 7.0 Gy by whole-body exposure in groups of 10 at a dose rate of 0.78Gy/min using a Therapax X-RAD 320 system equipped with a 320-kV highstability X-ray generator, metal ceramic X-ray tube, variable x-ray beamcollimator and filter (Precision X-ray Incorporated, East Haven, Conn.).Dose levels were selected based on prior studies conducted with the samestrain of mice indicating the LD_(50/30) was between 6.5 and 7.0 Gy(data not shown).

Drug Formulation and Administration.

The appropriate volume of concentrated stock solutions were diluted withice cold saline to prepare dosing solutions of 0.2 mg/ml anti-murineMASP-2 antibody (mAbM11) or 0.5 mg/ml anti-human MASP-2 antibody (mAbH6)according to protocol. Administration of anti-MASP-2 antibody mAbM11 andmAbH6 was via IP injection using a 25-gauge needle base on animal weightto deliver 1 mg/kg mAbM11, 5 mg/kg mAbH6, or saline vehicle.

Study Design.

Mice were randomly assigned to the groups as described in Table 8. Bodyweight and temperature were measured and recorded daily. Mice in Groups7, 11 and 13 were sacrificed at post-irradiation day 7 and bloodcollected by cardiac puncture under deep anesthesia. Surviving animalsat post-irradiation day 30 were sacrificed in the same manner and bloodcollected. Plasma was prepared from collected blood samples according toprotocol and returned to Sponsor for analysis.

TABLE 8 Study Groups Group Irradiation ID N Level (Gy) Treatment DoseSchedule  1 20 6.5 Vehicle 18 hr prior to irradiation, 2 hr postirradiation, weekly booster  2 20 6.5 anti-murine 18 hr prior toirradiation MASP-2 ab only (mAbM11)  3 20 6.5 anti-murine 18 hr prior toirradiation, 2 MASP-2 ab hr post irradiation, weekly (mAbM11) booster  420 6.5 anti-murine 2 hr post irradiation, MASP-2 ab weekly booster(mAbM11)  5 20 6.5 anti-human 18 hr prior to irradiation, 2 MASP-2 ab hrpost irradiation, weekly (mAbH6) booster  6 20 7.0 Vehicle 18 hr priorto irradiation, 2 hr post irradiation, weekly booster  7 5 7.0 Vehicle 2hr post irradiation only  8 20 7.0 anti-murine 18 hr prior toirradiation MASP-2 ab only (mAbM11)  9 20 7.0 anti-murine 18 hr prior toirradiation, 2 MASP-2 ab hr post irradiation, weekly (mAbM11) booster 1020 7.0 anti-murine 2 hr post irradiation, MASP-2 ab weekly booster(mAbM11) 11 5 7.0 anti-murine 2 hr post irradiation only MASP-2 ab(mAbM11) 12 20 7.0 anti-human 18 hr prior to irradiation, 2 MASP-2 ab hrpost irradiation, weekly (mAbH6) booster 13 5 None None None

Statistical Analysis.

Kaplan-Meier survival curves were generated and used to compare meansurvival time between treatment groups using log-Rank and Wilcoxonmethods. Averages with standard deviations, or means with standard errorof the mean are reported. Statistical comparisons were made using atwo-tailed unpaired t-test between controlled irradiated animals andindividual treatment groups.

Results

Kaplan-Meier survival plots for 7.0 and 6.5 Gy exposure groups areprovided in FIGS. 32A and 32B, respectively, and summarized below inTable 9. Overall, treatment with anti-murine MASP-2 ab (mAbM11)pre-irradiation increased the survival of irradiated mice compared tovehicle treated irradiated control animals at both 6.5 (20% increase)and 7.0 Gy (30% increase) exposure levels. At the 6.5 Gy exposure level,post-irradiation treatment with anti-murine MASP-2 ab resulted in amodest increase in survival (15%) compared to vehicle control irradiatedanimals.

In comparison, all treated animals at the 7.0 Gy exposure level showedan increase in survival compared to vehicle treated irradiated controlanimals. The greatest change in survival occurred in animals receivingmAbH6, with a 45% increase compared to control animals. Further, at the7.0 Gy exposure level, mortalities in the mAbH6 treated group firstoccurred at post-irradiation day 15 compared to post-irradiation day 8for vehicle treated irradiated control animals, an increase of 7 daysover control animals. Mean time to mortality for mice receiving mAbH6(27.3±1.3 days) was significantly increased (p=0.0087) compared tocontrol animals (20.7±2.0 days) at the 7.0 Gy exposure level.

The percent change in body weight compared to pre-irradiation day (day−1) was recorded throughout the study. A transient weight loss occurredin all irradiated animals, with no evidence of differential changes dueto mAbM11 or mAbH6 treatment compared to controls (data not shown). Atstudy termination, all surviving animals showed an increase in bodyweight from starting (day −1) body weight.

TABLE 9 Survival rates of test animals exposed to radiation Time toDeath First/Last Exposure Survival (Mean ± SEM, Death Test Group Level(%) Day) (Day) Control Irradiation 6.5 Gy 65% 24.0 ± 2.0   9/16 mAbM11pre- 6.5 Gy 85% 27.7 ± 1.5  13/17 exposure mAbM11 pre + 6.5 Gy 65% 24.0± 2.0   9/15 post-exposure mAbM11 post- 6.5 Gy 80% 26.3 ± 1.9   9/13exposure mAbH6 pre + 6.5 Gy 65% 24.6 ± 1.9   9/19 post-exposure Controlirraditation 7.0 Gy 35% 20.7 ± 2.0   8/17 mAbM11 pre- 7.0 Gy 65% 23.0 ±2.3   7/13 exposure mAbM11 pre + 7.0 Gy 55% 21.6 ± 2.2   7/16post-exposure mAbM11 post- 7.0 Gy 70% 24.3 ± 2.1   9/14 exposure mAbH6pre + 7.0 Gy 80% 27.3 ± 1.3* 15/20 post-exposure *p = 0.0087 bytwo-tailed unpaired t-test between controlled irradiated animals andtreatment group at the same irradiation exposure level.

Discussion

Acute radiation syndrome consists of three defined subsyndromes:hematopoietic, gastrointestinal, and cerebrovascular. The syndromeobserved depends on the radiation dose, with the hematopoietic effectsobserved in humans with significant partial or whole-body radiationexposures exceeding 1 Gy. The hematopoietic syndrome is characterized bysevere depression of bone-marrow function leading to pancytopenia withchanges in blood counts, red and white blood cells, and plateletsoccurring concomitant with damage to the immune system. As nadir occurs,with few neutrophils and platelets present in peripheral blood,neutropenia, fever, complications of sepsis and uncontrollablehemorrhage lead to death.

In the present study, administration of mAbH6 was found to increasesurvivability of whole-body x-ray irradiation in Swiss-Webster male miceirradiated at 7.0 Gy. Notably, at the 7.0 Gy exposure level, 80% of theanimals receiving mAbH6 survived to 30 days compared to 35% of vehicletreated control irradiated animals. Importantly, the first day of deathin this treated group did not occur until post-irradiation day 15, a7-day increase over that observed in vehicle treated control irradiatedanimals. Curiously, at the lower X-ray exposure (6.5 Gy), administrationof mAbH6 did not appear to impact survivability or delay in mortalitycompared to vehicle treated control irradiated animals. There could bemultiple reasons for this difference in response between exposurelevels, although verification of any hypothesis may require additionalstudies, including interim sample collection for microbiological cultureand hematological parameters. One explanation may simply be that thenumber of animals assigned to groups may have precluded seeing anysubtle treatment-related differences. For example, with groups sizes ofn=20, the difference in survival between 65% (mAbH6 at 6.5 Gy exposure)and 80% (mAbH6 at 7.0 Gy exposure) is 3 animals. On the other hand, thedifference between 35% (vehicle control at 7.0 Gy exposure) and 80%(mAbH6 at 7.0 Gy exposure) is 9 animals, and provides sound evidence ofa treatment-related difference.

These results demonstrate that anti-MASP-2 antibodies are effective intreating a mammalian subject at risk for, or suffering from thedetrimental effects of acute radiation syndrome.

Example 30

This Example demonstrates that MASP-2 deficient mice are protected fromNeisseria meningitidis induced mortality after infection with either N.meningitidis serogroup A or Neisseria meningitidis serogroup B.

Methods:

MASP-2 knockout mice (MASP-2 KO mice) were generated as described inExample 1. 10-week-old MASP-2 KO mice (n=10) and wild-type (WT) C57/BL6mice (n=10) were inoculated by intraperitoneal (i.p.) injection with adosage of 2.6×10⁷ CFU of Neisseria meningitidis serogroup A Z2491 in avolume of 100 μl. The infective dose was administered to mice inconjunction with iron dextran at a final concentration of 400 mg/kg.Survival of the mice after infection was monitored over a 72-hour timeperiod.

In a separate experiment, 10-week-old MASP-2 KO mice (n=10) andwild-type C57/BL6 mice (n=10) were inoculated by i.p. injection with adosage of 6×10⁶ CFU of Neisseria meningitidis serogroup B strain MC58 ina volume of 100 μl. The infective dose was administered to mice inconjunction with iron dextran at a final dose of 400 mg/kg. Survival ofthe mice after infection was monitored over a 72-hour time period. Anillness score was also determined for the WT and MASP-2 KO mice duringthe 72-hour time period after infection, based on the illness scoringparameters described below in TABLE 10, which is based on the scheme ofFransen et al. (2010) with slight modifications.

TABLE 10 Illness Scoring associated with clinical signs in infected miceSigns Score Normal 0 Slightly ruffled fur 1 Ruffled fur, slow and stickyeyes 2 Ruffled fur, lethargic and eyes shut 3 Very sick and no movementafter 4 stimulation Dead 5

Blood samples were taken from the mice at hourly intervals afterinfection and analyzed to determine the serum level (log cfu/mL) of N.meningitidis in order to verify infection and determine the rate ofclearance of the bacteria from the serum.

Results:

FIG. 33 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of MASP-2 KO and WT mice after administration of an infectivedose of 2.6×10⁷ cfu of N. meningitidis serogroup A Z2491. As shown inFIG. 33, 100% of the MASP-2 KO mice survived throughout the 72-hourperiod after infection. In contrast, only 80% of the WT mice (p=0.012)were still alive 24 hours after infection, and only 50% of the WT micewere still alive at 72 hours after infection. These results demonstratethat MASP-2-deficient mice are protected from N. meningitidis serogroupA Z2491-induced mortality.

FIG. 34 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of MASP-2 KO and WT mice after administration of an infectivedose of 6×10⁶ cfu of N. meningitidis serogroup B strain MC58. As shownin FIG. 34, 90% of the MASP-2 KO mice survived throughout the 72-hourperiod after infection. In contrast, only 20% of the WT mice (p=0.0022)were still alive 24 hours after infection. These results demonstratethat MASP-2-deficient mice are protected from N. meningitidis serogroupB strain MC58-induced mortality.

FIG. 35 graphically illustrates the log cfu/mL of N. meningitidisserogroup B strain MC58 recovered at different time points in bloodsamples taken from the MASP-2 KO and WT mice after i.p. infection with6×10⁶ cfu of N. meningitidis serogroup B strain MC58 (n=3 at differenttime points for both groups of mice). The results are expressed asMeans±SEM. As shown in FIG. 35, in WT mice the level of N. meningitidisin the blood reached a peak of about 6.0 log cfu/mL at 24 hours afterinfection and dropped to about 4.0 log cfu/mL by 36 hours afterinfection. In contrast, in the MASP-2 KO mice, the level of N.meningitidis reached a peak of about 4.0 log cfu/mL at 12 hours afterinfection and dropped to about 1.0 log cfu/mL by 36 hours afterinfection (the symbol “*” indicates p<0.05; the symbol “**” indicatesp=0.00⁴³). These results demonstrate that although the MASP-2 KO micewere infected with the same dose of N. meningitidis serogroup B strainMC58 as the WT mice, the MASP-2 KO mice have enhanced clearance ofbacteraemia as compared to WT.

FIG. 36 graphically illustrates the average illness score of MASP-2 KOand WT mice at 3, 6, 12 and 24 hours after infection with 6×10⁶ cfu ofN. meningitidis serogroup B strain MC58. As shown in FIG. 36, theMASP-2-deficient mice showed high resistance to the infection, with muchlower illness scores at 6 hours (symbol “*” indicates p=0.0411), 12hours (symbol “**” indicates p=0.00⁴⁹) and 24 hours (symbol “***”indicates p=0.0049) after infection, as compared to WT mice. The resultsin FIG. 36 are expressed as means±SEM.

In summary, the results in this Example demonstrate thatMASP-2-deficient mice are protected from Neisseria meningitides-inducedmortality after infection with either N. meningitidis serogroup A or N.meningitidis serogroup B.

Example 31

This Example demonstrates that the administration of anti-MASP-2antibody after infection with N. meningitidis increases the survival ofmice infected with N. meningitidis.

Background/Rationale:

As described in Example 10, rat MASP-2 protein was utilized to pan a Fabphage display library, from which Fab2 #11 was identified as afunctionally active antibody. Full-length antibodies of the rat IgG2cand mouse IgG2a isotypes were generated from Fab2 #11. The full-lengthanti-MASP-2 antibody of the mouse IgG2a isotype was characterized forpharmacodynamic parameters (as described in Example 19).

In this Example, the mouse anti-MASP-2 full-length antibody derived fromFab2 #11 was analyzed in the mouse model of N. meningitidis infection.

Methods:

The mouse IgG2a full-length anti-MASP-2 antibody isotype derived fromFab2 #11, generated as described above, was tested in the mouse model ofN. meningitidis infection as follows.

Administration of Mouse-Anti-MASP-2 Monoclonal Antibodies (MoAb) afterInfection

9-week-old C57/BL6 Charles River mice were treated with inhibitory mouseanti-MASP-2 antibody (1.0 mg/kg) (n=12) or control isotype antibody(n=10) at 3 hours after i.p. injection with a high dose (4×10⁶ cfu) ofN. meningitidis serogroup B strain MC58.

Results:

FIG. 37 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of mice after administration of an infective dose of 4×10⁶ cfuof N. meningitidis serogroup B strain MC58, followed by administration 3hours post-infection of either inhibitory anti-MASP-2 antibody (1.0mg/kg) or control isotype antibody. As shown in FIG. 37, 90% of the micetreated with anti-MASP-2 antibody survived throughout the 72-hour periodafter infection. In contrast, only 50% of the mice treated with isotypecontrol antibody survived throughout the 72-hour period after infection.The symbol “*” indicates p=0.0301, as determined by comparison of thetwo survival curves.

These results demonstrate that administration of anti-MASP-2 antibody iseffective to treat and improve survival in subjects infected with N.meningitidis.

As demonstrated herein, the use of anti-MASP-2 antibody in the treatmentof a subject infected with N. meningitidis is effective whenadministered within 3 hours post-infection, and is expected to beeffective within 24 hours to 48 hours after infection. Meningococcaldisease (either meningococcemia or meningitis) is a medical emergency,and therapy will typically be initiated immediately if meningococcaldisease is suspected (i.e., before N. meningitidis is positivelyidentified as the etiological agent).

In view of the results in the MASP-2 KO mouse demonstrated in EXAMPLE30, it is believed that administration of anti-MASP-2 antibody prior toinfection with N. meningitidis would also be effective to prevent orameliorate the severity of infection.

Example 32

This Example demonstrates that administration of anti-MASP-2 antibody iseffective to treat N. meningitidis infection in human serum.

Rationale:

Patients with decreased serum levels of functional MBL display increasedsusceptibility to recurrent bacterial and fungal infections (Kilpatricket al., Biochim Biophys Acta 1572:401-413 (2002)). It is known that N.meningitidis is recognized by MBL, and it has been shown thatMBL-deficient sera do not lyse Neisseria.

In view of the results described in Examples 30 and 31, a series ofexperiments were carried out to determine the efficacy of administrationof anti-MASP-2 antibody to treat N. meningitidis infection incomplement-deficient and control human sera. Experiments were carriedout in a high concentration of serum (20%) in order to preserve thecomplement pathway.

Methods:

1. Serum Bactericidal Activity in Various Complement-Deficient HumanSera and in Human Sera Treated with Human Anti-MASP-2 Antibody

The following complement-deficient human sera and control human serawere used in this experiment:

TABLE 11 Human sera samples tested (as shown in FIG. 38) Sample Serumtype A Normal human sera (NHS) + human anti-MASP-2 Ab B NHS + isotypecontrol Ab C MBL −/− human serum D NHS E Heat-Inactivated (HI) NHS

A recombinant antibody against human MASP-2 was isolated from aCombinatorial Antibody Library (Knappik, A., et al., J. Mol. Biol.296:57-86 (2000)), using recombinant human MASP-2A as an antigen (Chen,C. B. and Wallis, J. Biol. Chem. 276:25894-25902 (2001)). An anti-humanscFv fragment that potently inhibited lectin pathway-mediated activationof C4 and C3 in human plasma (IC50˜20 nM) was identified and convertedto a full-length human IgG4 antibody.

N. meningitidis serogroup B-MC58 was incubated with the different serashow in TABLE 11, each at a serum concentration of 20%, with or withoutthe addition of inhibitory human anti-MASP-2 antibody (3 μg in 100 μltotal volume) at 37° C. with shaking. Samples were taken at thefollowing time points: 0-, 30-, 60- and 90-minute intervals, plated outand then viable counts were determined. Heat-inactivated human serum wasused as a negative control.

Results:

FIG. 38 graphically illustrates the log cfu/mL of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in thehuman sera samples shown in TABLE 11. TABLE 12 provides the Student'st-test results for FIG. 38.

TABLE 12 Student's t-test Results for FIG. 38 (time point 60 minutes)Mean Diff. Significant? (Log) P < 0.05? P value summary A vs B −0.3678Yes ***(0.0002) A vs C −1.1053 Yes ***(p < 0.0001) A vs D −0.2111 Yes **(0.0012) C vs D 1.9 Yes ***(p < 0.0001)

As shown in FIG. 38 and TABLE 12, complement-dependent killing of N.meningitidis in human 20% serum was significantly enhanced by theaddition of the human anti-MASP-2 inhibitory antibody.

2. Complement-Dependent Killing of N. meningitidis in 20% (v/v) MouseSera Deficient of MASP-2.

The following complement-deficient mouse sera and control mouse serawere used in this experiment:

TABLE 13 Mouse sera samples tested (as shown in FIG. 39) Sample SerumType A WT B MASP-2 -/- C MBL A/C -/- D WT heat-inactivated (HIS)

N. meningitidis serogroup B-MC58 was incubated with differentcomplement-deficient mouse sera, each at a serum concentration of 20%,at 37° C. with shaking. Samples were taken at the following time points:0-, 15-, 30-, 60-, 90- and 120-minute intervals, plated out and thenviable counts were determined. Heat-inactivated human serum was used asa negative control.

Results:

FIG. 39 graphically illustrates the log cfu/mL of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in themouse sera samples shown in TABLE 13. As shown in FIG. 39, the MASP-2−/− mouse sera have a higher level of bactericidal activity for N.meningitidis than WT mouse sera. The symbol “**” indicates p=0.0058, thesymbol “***” indicates p=0.001. TABLE 14 provides the Student's t-testresults for FIG. 39.

TABLE 14 Student's t-test Results for FIG. 39 Mean Diff. Significant?Comparison Time point (LOG) (p < 0.05)? P value summary A vs. B 60 min.0.39 yes  ** (0.0058) A vs. B 90 min. 0.6741 yes *** (0.001)

In summary, the results in this Example demonstrate that MASP-2 −/− serahas a higher level of bactericidal activity for N. meningitidis than WTsera.

Example 33

This Example demonstrates the inhibitory effect of MASP-2 deficiency onlysis of red blood cells from blood samples obtained from a mouse modelof paroxysmal nocturnal hemoglobinuria (PNH).

Background/Rationale:

Paroxysmal nocturnal hemoglobinuria (PNH), also referred to asMarchiafava-Micheli syndrome, is an acquired, potentiallylife-threatening disease of the blood, characterized bycomplement-induced intravascular hemolytic anemia. The hallmark of PNHis the chronic complement-mediated intravascular hemolysis that is aconsequence of unregulated activation of the alternative pathway ofcomplement due to the absence of the complement regulators CD55 and CD59on PNH erythrocytes, with subsequent hemoglobinuria and anemia.Lindorfer, M. A., et al., Blood 115(11) (2010), Risitano, A. M,Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011). Anemia in PNH isdue to destruction of red blood cells in the bloodstream. Symptoms ofPNH include red urine, due to appearance of hemoglobin in the urine,back pain, fatigue, shortness of breath and thrombosis. PNH may developon its own, referred to as “primary PNH” or in the context of other bonemarrow disorders such as aplastic anemia, referred to as “secondaryPNH”. Treatment for PNH includes blood transfusion for anemia,anticoagulation for thrombosis and the use of the monoclonal antibodyeculizumab (Soliris®), which protects blood cells against immunedestruction by inhibiting the complement system (Hillmen P. et al., N.Engl. J. Med. 350(6):552-9 (2004)). Eculizumab (Soliris®) is a humanizedmonoclonal antibody that targets the complement component C5, blockingits cleavage by C5 convertases, thereby preventing the production of C5aand the assembly of the MAC. Treatment of PNH patients with eculizumabhas resulted in a reduction of intravascular hemolysis, as measured bylactate dehydrogenase (LDH), leading to hemoglobin stabilization andtransfusion independence in about half of the patients (Hillmen P, etal., Mini-Reviews in Medicinal Chemistry, vol 11(6) (2011)). Whilenearly all patients undergoing therapy with eculizumab achieve normal oralmost normal LDH levels (due to control of intravascular hemolysis),only about one third of the patients reach a hemoglobin value above 11gr/dL, and the remaining patients on eculizumab continue to exhibitmoderate to severe (i.e.,transfusion-dependent) anemia, in about equalproportions (Risitano A. M. et al., Blood 113:4094-100 (2009)). Asdescribed in Risitano et al., Mini-Reviews in Medicinal Chemistry11:528-535 (2011), it was demonstrated that PNH patients on eculizumabcontained C3 fragments bound to a substantial portion of their PNHerythrocytes (while untreated patients did not), leading to theconclusion that membrane-bound C3 fragments work as opsonins on PNHerythrocytes, resulting in their entrapment in the reticuloendothelialcells through specific C3 receptors and subsequent extravascularhemolysis. Therefore, therapeutic strategies in addition to the use ofeculizumab are needed for those patients developing C3 fragment-mediatedextravascular hemolysis because they continue to require red celltransfusions.

This Example describes methods to assess the effect of MASP-2-deficientserum and serum treated with MASP-2 inhibitory agent on lysis of redblood cells from blood samples obtained from a mouse model of PNH anddemonstrates the efficacy of MASP-2 inhibition to treat subjectssuffering from PNH, and also supports the use of inhibitors of MASP-2 toameliorate the effects of C3 fragment-mediated extravascular hemolysisin PNH subjects undergoing therapy with a C5 inhibitor such aseculizumab.

Methods:

PNH Animal Model:

Blood samples were obtained from gene-targeted mice with deficiencies ofCrry and C3 (Crry/C3−/−) and CD55/CD59-deficient mice. These mice aremissing the respective surface complement regulators and theirerythrocytes are, therefore, susceptible to spontaneous complementautolysis as are PNH human blood cells.

In order to sensitize these erythrocytes even more, these cells wereused with and without coating by mannan and then tested for hemolysis inWT C56/BL6 plasma, MBL null plasma, MASP-2 −/− plasma, human NHS, humanMBL −/− plasma, and NHS treated with human anti-MASP-2 antibody.

1. Hemolysis Assay of Crry/C3 and CD55/CD59 Double-Deficient MarineErythrocytes in MASP-2-Deficient/Depleted Sera and Controls Day 1.Preparation of Murine RBC (±Mannan Coating)

Materials included:

fresh mouse blood, BBS/Mg^(2+/)Ca²⁺ (4.4 mM barbituric acid, 1.8 mMsodium barbitone, 145 mM NaCl, pH7.4, 5 mM Mg²⁺, 5 mM Ca²⁺), chromiumchloride, CrCl₃.6H₂0 (0.5 mg/mL in BBS/Mg2+/Ca2+) and mannan, 100 μg/mLin BBS/Mg2+/Ca2+.

Whole blood (2 mL) was spun down for 1-2 min at 2000×g in a refrigeratedcentrifuge at 4′C. The plasma and buffy coat were aspirated off. Thesample was then washed three times by re-suspending the RBC pellet in 2mL ice-cold BBS/gelatin/Mg2+/Ca2+ and repeating centrifugation step.After the third wash, the pellet was re-suspended in 4 mL BBS/Mg2+/Ca2+.A 2 mL aliquot of the RBC was set aside as an uncoated control. To theremaining 2 mL, 2 mL CrCl3 and 2 mL mannan were added and the sample wasincubated with gentle mixing at room temperature for 5 minutes. Thereaction was terminated by adding 7.5 mL BBS/gelatin/Mg2+/Ca2+. Thesample was spun down as above, re-suspended in 2 mLBBS/gelatin/Mg2+/Ca2+ and washed a further two times as above, thenstored at 4′C.

Day 2. Hemolysis Assay

Materials included BBS/gelatin/Mg²⁺/Ca²⁺ (as above), test sera, 96-wellround-bottomed and flat-bottomed plates and a spectrophotometer thatreads 96-well plates at 410-414 nm.

The concentration of the RBC was first determined and the cells wereadjusted to 10⁹/mL, and stored at this concentration. Before use, theassay buffer was diluted to 108/mL, and then 100 ul per well was used.Hemolysis was measured at 410-414 nm (allowing for greater sensitivitythen 541 nm). Dilutions of test sera were prepared in ice-coldBBS/gelatin/Mg2+/Ca2+. 100 μl of each serum dilution was pipetted intoround-bottomed plate (see plate layout). 100 μl of appropriately dilutedRBC preparation was added (i.e., 10⁸/mL) (see plate layout), incubatedat 37° C. for about 1 hour, and observed for lysis. (The plates may bephotographed at this point.) The plate was then spun down at maximumspeed for 5 minutes. 100 μl was aspirated of the fluid-phase,transferred to flat-bottom plates, and the OD was recorded at 410-414nm. The RBC pellets were retained (these can be subsequently lysed withwater to obtain an inverse result).

Experiment #1

Fresh blood was obtained from CD55/CD59 double-deficient mice and bloodof Crry/C3 double-deficient mice and erythrocytes were prepared asdescribed in detail in the above protocol. The cells were split and halfof the cells were coated with mannan and the other half were leftuntreated, adjusting the final concentration to 1×108 per mL, of which100 μl was used in the hemolysis assay, which was carried out asdescribed above.

Results of Experiment #1: The Lectin Pathway is Involved in ErythrocyteIysis in the PNH Animal Model

In an initial experiment, it was determined that non-coated WT mouseerythrocytes were not lysed in any mouse serum. It was furtherdetermined that mannan-coated Crry−/− mouse erythrocytes were slowlylysed (more than 3 hours at 37 degrees) in WT mouse serum, but they werenot lysed in MBL null serum. (Data not shown).

It was determined that mannan-coated Crry−/− mouse erythrocytes wererapidly lysed in human serum but not in heat-inactivated NHS.Importantly, mannan-coated Crry−/− mouse erythrocytes were lysed in NHSdiluted down to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320 and 1/640dilutions all lysed). (Data not shown). In this dilution, thealternative pathway does not work (AP functional activity issignificantly reduced below 8% serum concentration).

Conclusions from Experiment #1

Mannan-coated Crry−/− mouse erythrocytes are very well lysed in highlydiluted human serum with MBL but not in that without MBL. The efficientlysis in every serum concentration tested implies that the alternativepathway is not involved or needed for this lysis. The inability ofMBL-deficient mouse serum and human serum to lyse the mannan-coatedCrry−/− mouse erythrocytes indicates that the classical pathway also hasnothing to do with the lysis observed. As lectin pathway recognitionmolecules are required (i.e., MBL), this lysis is mediated by the lectinpathway.

Experiment #2

Fresh blood was obtained from the Crry/C3 and CD55/CD59 double-deficientmice and mannan-coated Crry−/− mouse erythrocytes were analyzed in thehaemolysis assay as described above in the presence of the followinghuman serum: MBL null; WT; NHS pretreated with human anti-MASP-2antibody; and heat-inactivated NHS as a control.

Results of Experiment #2: MASP-2 Inhibitors Prevent Erythrocyte Lysis inthe PNH Animal Model

With the Mannan-coated Crry−/− mouse erythrocytes, NHS was incubated inthe dilutions diluted down to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320 and1/640), human MBL−/− serum, NHS pretreated with anti-MASP-2 mAb, andheat-inactivated NHS as a control.

The ELISA microtiter plate was spun down and the non-lysed erythrocyteswere collected on the bottom of the round-well plate. The supernatant ofeach well was collected and the amount of hemoglobin released from thelysed erythrocytes was measured by reading the OD415 nm in an ELISAreader.

In the control heat-inactivated NHS (negative control), as expected, nolysis was observed. MBL−/− human serum lysed mannan-coated mouseerythrocytes at ⅛ and 1/16 dilutions. Anti-MASP-2-antibody-pretreatedNHS lysed mannan-coated mouse erythrocytes at ⅛ and 1/16 dilutions whileWT human serum lysed mannan-coated mouse erythrocytes down to dilutionsof 1/32.

FIG. 40 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed mouse erythrocytes (Cryy/C3−/−) into the supernatantmeasured by photometry) of mannan-coated murine erythrocytes by humanserum over a range of serum concentrations in serum fromheat-inactivated (HI) NHS, MBL−/−, NHS pretreated with anti-MASP-2antibody, and NHS control.

From the results shown in FIG. 40, it is demonstrated that MASP-2inhibition with anti-MASP-2 antibody significantly shifted the CH₅₀ andinhibited complement-mediated lysis of sensitized erythrocytes withdeficient protection from autologous complement activation.

Experiment #3

Fresh blood obtained from the Crry/C3 and CD55/CD59 double-deficientmice in non-coated Crry−/− mouse erythrocytes was analyzed in thehemolysis assay as described above in the presence of the followingserum: MBL −/−; WT sera; NHS pretreated with human anti-MASP-2 antibodyand heat-inactivated NHS as a control.

Results:

FIG. 41 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed WT mouse erythrocytes into the supernatant measured byphotometry) of non-coated murine erythrocytes by human serum over arange of serum concentrations in serum from heat inactivated (HI) NHS,MBL−/−, NHS pretreated with anti-MASP-2 antibody, and NHS control. Asshown in FIG. 41, it is demonstrated that inhibiting MASP-2 inhibitscomplement-mediated lysis of non-sensitized WT mouse erythrocytes.

FIG. 42 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed mouse erythrocytes (CD55/59 −/−) into the supernatantmeasured by photometry) of non-coated murine erythrocytes by human serumover a range of serum concentration in serum from heat-inactivated (HI)NHS, MBL−/−, NHS pretreated with anti-MASP-2 antibody, and NHS control.

TABLE 12 CH₅₀ values expressed as serum concentrations Serum WT CD55/59-/- Heat-inactivated NHS No lysis No lysis MBL AO/XX donor 7.2%  2.1%(MBL deficient) NHS + anti-MASP-2 5.4%  1.5% antibody NHS 3.1% 0.73%Note: “CH₅₀” is the point at which complement mediated hemolysis reaches50%.

In summary, the results in this Example demonstrate that inhibitingMASP-2 inhibits complement-mediated lysis of sensitized andnon-sensitized erythrocytes with deficient protection from autologouscomplement activation. Therefore, MASP-2 inhibitors may be used to treatsubjects suffering from PNH, and may also be used to ameliorate (i.e.,inhibit, prevent or reduce the severity of) extravascular hemolysis inPNH patients undergoing treatment with a C5 inhibitor such as eculizumab(Soliris®).

Example 34

This Example describes a follow on study to the study described above inExample 29, providing further evidence confirming that a MASP-2inhibitor, such as a MASP-2 antibody, is effective for the treatment ofradiation exposure and/or for the treatment, amelioration or preventionof acute radiation syndrome.

Rationale:

In the initial study described in Example 29, it was demonstrated thatpre-irradiation treatment with an anti-MASP-2 antibody in mice increasedthe survival of irradiated mice as compared to vehicle treatedirradiated control animals at both 6.5 Gy and 7.0 Gy exposure levels. Itwas further demonstrated in Example 29 that at the 6.5 Gy exposurelevel, post-irradiation treatment with anti-MASP-2 antibody resulted ina modest increase in survival as compared to vehicle control irradiatedanimals. This Example describes a second radiation study that wascarried out to confirm the results of the first study.

Methods: Design of Study A:

Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy).The effect of anti-MASP-2 antibody therapy (mAbH6 5 mg/kg), administered18 hours before and 2 hours after radiation exposure, and weeklythereafter, on mortality was assessed.

Results of Study A:

As shown in FIG. 43, it was determined that administration of theanti-MASP-2 antibody mAbH6 increased survival in mice exposed to 8.0 Gy,with an adjusted median survival rate increased from 4 to 6 days ascompared to mice that received vehicle control, and a mortality reducedby 12% when compared to mice that received vehicle control (log-ranktest, p=0.040).

Design of Study B:

Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy) inthe following groups (I: vehicle) saline control; (II: low) anti-MASP-2antibody mAbH6 (5 mg/kg) administered 18 hours before irradiation and 2hours after irradiation; (III: high) mAbH6 (10 mg/kg) administered 18hours before irradiation and 2 hours post irradiation; and (IV:highpost) mAbH6 (10 mg/kg) administered 2 hours post irradiation only.

Results of Study B:

Administration of anti-MASP-2 antibody pre- and post-irradiationadjusted the mean survival from 4 to 5 days in comparison to animalsthat received vehicle control. Mortality in the anti-MASP-2antibody-treated mice was reduced by 6-12% in comparison to vehiclecontrol mice. It is further noted that no significant detrimentaltreatment effects were observed (data not shown).

In summary, the results shown in this Example are consistent with theresults shown in Example 29 and further demonstrate that anti-MASP-2antibodies are effective in treating a mammalian subject at risk for, orsuffering from the detrimental effects of acute radiation syndrome.

Example 35

This study investigates the effect of MASP-2-deficiency in a mouse modelof LPS (lipopolysaccharide)-induced thrombosis.

Rationale:

Hemolytic uremic syndrome (HUS), which is caused by Shigatoxin-producing E. coli infection, is the leading cause of acute renalfailure in children. In this Example, a Schwartzman model of LPS-inducedthrombosis (microvascular coagulation) was carried out in MASP-2−/− (KO)mice to determine whether MASP-2 inhibition is effective to inhibit orprevent the formation of intravascular thrombi.

Methods:

MASP-2−/− (n=9) and WT (n=10) mice were analyzed in a Schwarztman modelof LPS-induced thrombosis (microvascular coagulation). Mice wereadministered Serratia LPS and thrombus formation was monitored overtime. A comparison of the incidence of microthromi and LPS-inducedmicrovascular coagulation was carried out.

Results:

Notably, all MASP-2 −/− mice tested (9/9) did not form intravascularthrombi after Serratia LPS administration. In contrast, microthrombiwere detected in 7 of 10 of the WT mice tested in parallel (p=0.0031,Fischer's exact). As shown in FIG. 44, the time to onset ofmicrovascular occlusion following LPS infection was measured inMASP-2−/− and WT mice, showing the percentage of WT mice with thrombusformation measured over 60 minutes, with thrombus formation detected asearly as about 15 minutes. Up to 80% of the WT mice demonstratedthrombus formation at 60 minutes. In contrast, as shown in FIG. 44, noneof the MASP-2 −/− had thrombus formation at 60 minutes (log rank:p=0.0005).

These results demonstrate that MASP-2 inhibition is protective againstthe development of intravascular thrombi in an HUS model.

Example 36

This Example describes the effect of anti-MASP-2 antibodies in a mousemodel of HUS using intraperitoneal co-injection of purified Shiga toxin2 (STX2) plus LPS.

Background

A mouse model of HUS was developed using intraperitoneal co-injection ofpurified Shiga toxin 2 (STX2) plus LPS. Biochemical and microarrayanalysis of mouse kidneys revealed the STX2 plus LPS challenge to bedistinct from the effects of either agent alone. Blood and serumanalysis of these mice showed neutrophilia, thrombocytopenia, red cellhemolysis, and increased serum creatinine and blood urea nitrogen. Inaddition, histologic analysis and electron microscopy of mouse kidneysdemonstrated glomerular fibrin deposition, red cell congestion,microthrombi formation, and glomerular ultrastructural changes. It wasestablished that this model of HUS induces all clinical symptoms ofhuman HUS pathology in C57BL/6 mice including thrombocytopenia,hemolytic anemia, and renal failure that define the human disease. (J.Immunol 187(1): 172-80 (2011))

Methods:

C57BL/6 female mice that weighed between 18 to 20 g were purchased fromCharles River Laboratories and divided in to 2 groups (5 mice in eachgroup). One group of mice was pretreated by intraperitoneal (i.p.)injection with the recombinant anti-MASP-2 antibody mAbM11 (100 μg permouse; corresponding to a final concentration of 5 mg/kg body weight)diluted in a total volume of 150 μl saline. The control group receivedsaline without any antibody. Six hours after i.p injection ofanti-MASP-2 antibody mAbM11, all mice received a combined i.p. injectionof a sublethal dose (3 μg/animal; corresponding to 150 μg/kg bodyweight) of LPS of Serratia marcescens (L6136; Sigma-Aldrich, St. Louis,Mo.) and a dose of 4.5 ng/animal (corresponding to 225 ng/kg) of STX2(two times the LD50 dose) in a total volume of 150 μl. Saline injectionwas used for control.

Survival of the mice was monitored every 6 hours after dosing. Mice wereculled as soon as they reached the lethargic stage of HUS pathology.After 36 hours, all mice were culled and both kidneys were removed forimmunohistochemistry and scanning electron microscopy. Blood sampleswere taken at the end of the experiment by cardiac puncture. Serum wasseparated and kept frozen at −80° C. for measuring BUN and serumCreatinine levels in both treated and control groups.

Immunohistochemistry

One-third of each mouse kidney was fixed in 4% paraformaldehyde for 24h, processed, and embedded in paraffin. Three-micron-thick sections werecut and placed onto charged slides for subsequent staining with H & Estain.

Electron Microscopy

The middle section of the kidneys was cut into blocks of approximately 1to 2 mm³, and fixed overnight at 4° C. in 2.5% glutaraldehyde in 1×PBS.The fixed tissue subsequently was processed by the University ofLeicester Electron Microscopy Facility

Cryostat Sections

The other third of the kidneys was, cut into blocks approximately 1 to 2mm³ and snap frozen in liquid nitrogen and kept at −80° C. for cryostatsections and mRNA analysis.

Results:

FIG. 45 graphically illustrates the percent survival of saline-treatedcontrol mice (n=5) and anti-MASP-2 antibody-treated mice (n=5) in theSTX/LPS-induced model over time (hours). Notably, as shown in FIG. 45,all of the control mice died by 42 hours. In sharp contrast, 100% of theanti-MASP-2 antibody-treated mice survived throughout the time course ofthe experiment. Consistent with the results shown in FIG. 45, it wasobserved that all the untreated mice that either died or had to beculled with signs of severe disease had significant glomerular injuries,while the glomeruli of all anti-MASP-2-treated mice looked normal (datanot shown). These results demonstrate that MASP-2 inhibitors, such asanti-MASP-2 antibodies, may be used to treat subjects suffering from, orat risk for developing a thrombotic microangiopathy (TMA), such ashemolytic uremic syndrome (HUS), atypical HUS (aHUS), or thromboticthrombocytopenic purpura (TTP).

Example 37

This Example describes the effect of MASP-2 deficiency and MASP-2inhibition in a murine FITC-dextran/light induced endothelial cellinjury model of thrombosis.

Background/Rationale:

As demonstrated in Examples 35 and 36, MASP-2 deficiency (MASP-2 KO) andMASP-2 inhibition (via administration of an inhibitory MASP-2 antibody)protects mice in a model of typical HUS, wherease all control miceexposed to STX and LPS developed severe HUS and became moribund or diedwithin 48 hours. For example, as shown in FIG. 54, all mice treated witha MASP-2 inhibitory antibody and then exposed to STX and LPS survived(Fisher's exact p<0.01; N=5). Thus, anti-MASP-2 therapy protects mice inthis model of HUS.

The following experiments were carried out to analzye the effect ofMASP-2 deficiency and MASP-2 inhibition in a fluorescein isothiocyanate(FITC)-dextran-induced endothelial cell injury model of thromboticmicroangiopathy (TMA) in order to demonstrate further the benefit ofMASP-2 inhibitors for the treatment of HUS, aHUS, TTP, and TMA's withother etiologies.

Methods: Intravital Microscopy

Mice were prepared for intravital microscopy as described by Frommholdet al., BMC Immunology 12:56-68, 2011. Briefly, mice were anesthetizedwith intraperitoneal (i.p.) injection of ketamine (125 mg/kg bodyweight,Ketanest, Pfitzer GmbH, Karlsruhe, Germany) and xylazine (12.5 mg/kgbody weight; Rompun, Bayer, Leverkusen, Germany) and placed on a heatingpad to maintain body temperature at 37° C. Intravital microscopy wasconducted on an upright microscope (Leica, Wetzlar, Germany) with asaline immersion objective (SW 40/0.75 numerical aperture, Zeiss, Jena,Germany). To ease breathing, mice were intubated using PE 90 tubing(Becton Dickson and Company, Sparks, Md., USA). The left carotid arterywas cannuled with PE10 tubing (Becton Dickson and Company, Sparks, Md.,USA) for blood sampling and systemic monoclonal antibody (mAb)administration.

Cremaster Muscle Preparation

The surgical preparation of the cremaster muscle for intravitalmicroscopy was performed as described by Sperandio et al., Blood,97:3812-3819, 2001. Briefly, the scrotum was opened and the cremastermuscle mobilized. After longitudinal incision and spreading of themuscle over a cover glass, the epididymis and testis were moved andpinned to the side, giving full microscopic access to the cremastermuscle microcirculation. Cremaster muscle venules were recorded via aCCD camera (CF8/1; Kappa, Gleichen, Germany) on a Panasonic S-VHSrecorder. The cremaster muscle was superfused with thermo-controlled(35° C. bicarbonate-buffered saline) as previously described byFrommhold et al., BMC Immunology 12:56-68, 20112011.

Light Excitation FITC Dextran Injury Model

A controlled, light-dose-dependent vascular injury of the endothelium ofcremaster muscle venules and arterioles was induced by light excitationof phototoxic (FITC)-dextran (Cat. No. FD150S, Sigma Aldrich, Poole,U.K.). This procedure initiates localized thrombosis. As a phototoxicreagent, 60 μL of a 10% w/v solution of FITC-dextran was injectedthrough the left carotid artery access and allowed to spreadhomogenously throughout the circulating blood for 10 minutes. Afterselecting a well-perfused venule, halogen light of low to midrangeintensity (800-1500) was focused on the vessel of interest to induceFITC-dextran fluorescence and mild to moderate phototoxicity to theendothelial surface in order to stimulate thrombosis in a reproducible,controlled manner. The necessary phototoxic light intensity for theexcitation of FITC-dextran was generated using a halogen lamp (12V,100W, Zeiss, Oberkochen, Germany). The phototoxicity resulting fromlight-induced excitation of the fluorochrome requires a threshold oflight intensity and/or duration of illumination and is caused by eitherdirect heating of the endothelial surface or by generation of reactiveoxygen radicals as described by Steinbauer et al., Langenbecks Arch Surg385:290-298, 2000.

The intensity of the light applied to each vessel was measured foradjustment by a wavelength-correcting diode detector for low powermeasurements (Labmaster LM-2, Coherent, Auburn, USA). Off-line analysisof video scans was performed by means of a computer assistedmicrocirculation analyzing system (CAMAS, Dr. Zeintl, Heidelberg) andred blood cell velocity was measured as described by Zeintl et al., IntJMicrocirc Clin Exp, 8(3):293-302, 2000.

Application of Monoclonal Anti-Human MASP-2 Inhibitory Antibody (mAbH6)and Vehicle Control Prior to Induction of Thrombosis

Using a blinded study design, 9-week-old male C57BL/6 WT littermate micewere given i.p. injections of either the recombinant monoclonal humanMASP-2 antibody (mAbH6), an inhibitor of MASP-2 functional activity(given at a final concentration of 10 mg/kg body weight), or the samequantity of an isotype control antibody (without MASP-2 inhibitoryactivity) 16 hours before the phototoxic induction of thrombosis in thecremaster model of intravital microscopy. One hour prior to thrombosisinduction, a second dose of either mAbH6 or the control antibody wasgiven. MASP-2 knockout (KO) mice were also evaluated in this model.

mAbH6 (established against recombinant human MASP-2) is a potentinhibitor of human MASP-2 functional activity, which cross-reacts with,binds to and inhibits mouse MASP-2 but with lower affinity due to itsspecies specificity (data not shown). In order to compensate for thelower affinity of mAbH6 to mouse MASP-2, mAbH6 was given at a highconcentration (10 mg/kg body weight) to overcome the variation inspecies specificity, and the lesser affinity for mouse MASP-2, toprovide effective blockade of murine MASP-2 functional activity under invivo conditions.

In this blinded study, the time required for each individual venuoletested (selection criteria were by comparable diameters and blood flowvelocity) to fully occlude was recorded.

The percentage of mice with microvascular occlusion, the time of onset,and the time to occlusion were evaluated over a 60-minute observationperiod using intravital microscopy video recordings.

Results:

FIG. 46 graphically illustrates, as a function of time after injuryinduction, the percentage of mice with microvascular occlusion in theFITC/Dextran UV model after treatment with isotype control or humanMASP-2 antibody mAbH6 (10 mg/kg) dosed at 16 hours and 1 hour prior toinjection of FITC/Dextran. As shown in FIG. 46, 85% of the wild-typemice receiving the isotype control antibody occluded within 30 minutesor less, whereas only 19% of the wild-type mice pre-treated with thehuman MASP-2 antibody (mAbH6) occluded within the same time period, andthe time to occlusion was delayed in the mice that did eventuallyocclude in the human MASP-2 antibody-treated group. It is further notedthat three of the MASP-2 mAbH6 treated mice did not occlude at allwithin the 60-minute observation period (i.e., were protected fromthrombotic occlusion).

FIG. 47 graphically illustrates the occlusion time in minutes for micetreated with the human MASP-2 antibody (mAbH6) and the isotype controlantibody. The data are reported as scatter-dots with mean values(horizontal bars) and standard error bars (vertical bars). This figureshows the occlusion time in the mice where occlusion was observable.Thus, the three MASP-2 antibody-treated mice that did not occlude duringthe 60 minute observation period were not included in this analysis(there was no control treated mouse that did not occlude). Thestatistical test used for analysis was the unpaired t test; wherein thesymbol “*” indicates p=0.0129. As shown in FIG. 47, in the four MASP-2antibody (mAbH6)-treated mice that occluded, treatment with MASP-2antibody significantly increased the venous occlusion time in theFITC-dextran/light-induced endothelial cell injury model of thrombosiswith low light intensity (800-1500) as compared to the mice treated withthe isotype control antibody. The average of the full occlusion time ofthe isotype control was 19.75 minutes, while the average of the fullocclusion time for the MASP-2 antibody treated group was 32.5 minutes.

FIG. 48 graphically illustrates the time until occlusion in minutes forwild-type mice, MASP-2 KO mice, and wild-type mice pre-treated withhuman MASP-2 antibody (mAbH6) administered i.p. at 10 mg/kg 16 hoursbefore, and then administered again i.v.1 hour prior to the induction ofthrombosis in the FITC-dextran/light-induced endothelial cell injurymodel of thrombosis with low light intensity (800-1500). Only theanimals that occluded were included in FIG. 48; n=2 for wild-type micereceiving isotype control antibody; n=2 for MASP-2 KO; and n=4 forwild-type mice receiving human MASP-2 antibody (mAbH6). The symbol “*”indicates p<0.01. As shown in FIG. 48, MASP-2 deficiency and MASP-2inhibition (mAbH6 at 10 mg/kg) increased the venous occlusion time inthe FITC-dextran/light-induced endothelial cell injury model ofthrombosis with low light intensity (800-1500).

Conclusions:

The results in this Example further demonstrate that a MASP-2 inhibitoryagent that blocks the lectin pathway (e.g., antibodies that block MASP-2function), inhibits microvascular coagulation and thrombosis, thehallmarks of multiple microangiopathic disorders, in a mouse model ofTMA. Therefore, it is expected that administration of a MASP-2inhibitory agent, such as a MASP-2 inhibitory antibody, will be aneffective therapy in patients suffering from HUS, aHUS, TTP, or othermicroangiopathic disorders and provide protection from microvascularcoagulation and thrombosis.

Example 38

This Example describes a study demonstrating that human MASP-2inhibitory antibody (mAbH6) has no effect on platelet function inplatelet-rich human plasma.

Background/Rationale:

As described in Example 37, it was demonstrated that MASP-2 inhibitionwith human MASP-2 inhibitory antibody (mAbH6) increased the venousocclusion time in the FITC-dextran/light-induced endothelial cell injurymodel of thrombosis. The following experiment was carried out todetermine whether the MASP-2 inhibitory antibody (mAbH6) has an effecton platelet function.

Methods: The effect of human mAbH6 MASP-2 antibody was tested onADP-induced aggregation of platelets as follows. Human MASP-2 mAbH6 at aconcentration of either 1 μg/ml or 0.1 μg/ml was added in a 40 μLsolution to 360 μL of freshly prepared platelet-rich human plasma. Anisotype control antibody was used as the negative control. After addingthe antibodies to the plasma, platelet activation was induced by addingADP at a final concentration of 2 jM. The assay was started by stirringthe solutions with a small magnet in the 1 mL cuvette. Plateletaggregation was measured in a two-channel Chrono-log PlateletAggregometer Model 700 Whole Blood/Optical Lumi-Aggregometer.

Results:

The percent aggregation in the solutions was measured over a time periodof five minutes. The results are shown below in TABLE 13.

TABLE 13 Platelet Aggregation over a time period of five minutes. Slope(percent Amplitude aggregation Antibody (percent aggregation) over time)MASP-2 antibody (mAbH6) 46% 59   (1 μg/ml) Isotype control antibody 49%64   (1 μg/ml) MASP-2 antibody (mAbH6) 52% 63 (0.1 μg/ml) Isotypecontrol antibody 46% 59 (0.1 μg/ml)

As shown above in TABLE 13, no significant difference was observedbetween the aggregation of the ADP-induced platelets treated with thecontrol antibody or the MASP-2 mAbH6 antibody. These results demonstratethat the human MASP-2 antibody (mAbH6) has no effect on plateletfunction. Therefore, the results described in Example 37 demonstratingthat MASP-2 inhibition with human MASP-2 inhibitory antibody (mAbH6)increased the venous occlusion time in the FITC-dextran/light-inducedendothelial cell injury model of thrombosis, were not due to an effectof mAbH6 on platelet function. Thus, MASP-2 inhibition preventsthrombosis without directly impacting platelet function, revealing atherapeutic mechanism that is distinct from existing anti-thromboticagents.

Example 39

This Example describes the effect of MASP-2 inhibition on thrombusformation and vessel occlusion in a murine model of TMA.

Background/Rationale:

The lectin pathway plays a dominant role in activating the complementsystem in settings of endothelial cell stress or injury. This activationis amplified rapidly by the alternative pathway, which is dysregulatedin many patients presenting with aHUS. Preventing the activation ofMASP-2 and the lectin pathway is thus expected to halt the sequence ofenzymatic reactions that lead to the formation of the membrane attackcomplex, platelet activation, and leukocyte recruitment. This effectlimits tissue damage. In addition, MASP-2 has Factor Xa-like activityand cleaves prothrombin to form thrombin. This MASP-2-driven activationof the coagulation system may imbalance hemostasis and result in thepathology of TMA. Thus, inhibition of MASP-2 using a MASP-2 inhibitor,such as a MASP-2 inhibitory antibody that blocks activation of thecomplement and coagulation systems is expected to improve outcomes inaHUS and other TMA-related conditions.As described in Example 37, it was demonstrated that MASP-2 inhibitionwith human MASP-2 inhibitory antibody (mAbH6) increased the venousocclusion time in the FITC-dextran/light-induced endothelial cell injurymodel of thrombosis. In this model of TMA, mice were sensitized by IVinjection of FITC-dextran, followed by localized photo-activation of theFITC-dextran in the microvasculature of the mouse cremaster muscle(Thorlacius H et al., Eur J Clin. Invest 30(9):804-10, 2000; Agero etal., Toxicon 50(5):698-706, 2007).The following experiment was carried out to determine whether the MASP-2inhibitory antibody (mAbH6) has a dose-response effect on thrombusformation and vessel occlusion in a murine model of TMA.Methods: Localized thrombosis was induced by photo-activation offluorescein isothiocyanate-labeled dextran (FITC-dextran) in themicrovasculature of the cremaster muscle of C57 Bl/6 mice and intravitalmicroscopy was used to measure onset of thrombus formation and vesselocclusion using methods described in Example 37, with the followingmodifications. Groups of mice were dosed with mAbH6 (2 mg/kg, 10 mg/kgor 20 mg/kg) or isotype control antibody (20 mg/kg) were administered byintravenous (iv) injection one hour before TMA induction. The time toonset of thrombus formation and time to complete vessel occlusion wererecorded. Video playback analysis of intravital microscopy imagesrecorded over 30 to 60 minutes was used to evaluate vessel size, bloodflow velocity, light intensity, rate of onset of thrombus formation asequivalent of platelet adhesion, time to onset of thrombus formation,rate of total vessel occlusion and time until total vessel occlusion.Statistical analysis was conducted using SigmaPlot v12.0.

Results: Initiation of Thrombus Formation

FIG. 49 is a Kaplan-Meier plot showing the percentage of mice withthrombi as a function of time in FITC-Dextran induced thromboticmicroangiopathy in mice treated with increasing doses of human MASP-2inhibitory antibody (mAbH6 at 2 mg/kg, 10 mg/kg or 20 mg/kg) or anisotype control antibody. As shown in FIG. 49, initiation of thrombusformation was delayed in the mAbH6-treated mice in a dose-dependentmanner relative to the control-treated mice.FIG. 50 graphically illustrates the median time to onset (minutes) ofthrombus formation as a function of mAbH6 dose (*p<0.01 compared tocontrol). As shown in FIG. 50, the median time to onset of thrombusformation increased with increasing doses of mAbH6 from 6.8 minutes inthe control group to 17.7 minutes in the 20 mg/kg mAbH6 treated group(p<0.01). The underlying experimental data and statistical analysis areprovided in TABLES 14 and 15.The time to onset of thrombus formation in individual mice recordedbased on evaluation of the videographic recording is detailed below inTABLE 14.

TABLE 14 Time to Onset of Thrombus Formation After Light Dye-inducedInjury Control Treatment mAbH6 Treatment Control 2 mg/kg 10 mg/kg 20mg/kg Time to Onset 6.07 5.93 12.75 10.00 (minutes) 1.07 6.95 2.53 10.338.00 8.92 14.00 21.00 2.40 11.92 3.05 11.50 8.48 12.75 8.00 19.00 4.0012.53 8.17 10.37 4.00 15.83 22.65 7.83 11.70 16.37 6.83 50.67 21.75*15.00 32.25* 15.67 *vessels did not show onset during the indicatedobservation periodThe statistical analysis comparing time to onset of occlusion betweencontrol and mAbH6 treated animals is shown below in TABLE 15.

TABLE 15 Time to Onset: data from FITC Dex dose response study mAbH6mAbH6 mAbH6 Statistic Control (2 mg/kg) (10 mg/kg) (20 mg/kg) Number ofevents/number of 11/11 6/6 9/9 8/10 animals (%) (100%) (100%) (100%)(80.0%) Median time (minutes) (95% CI) 6.8 10.4 11.7 17.7 (2.4, 8.5)(5.9, 12.8) (2.5, 15.8) (10.0, 22.7) Wilcoxon p-value* 0.2364 0.19630.0016 Event = Time to onset observed Median (minutes) and its 95% CIwere based on Kaplan-Meier estimate NE = not estimable *p-values wereadjusted by Dunnett-Hsu multiple comparison

Microvascular Occlusion

FIG. 51 is a Kaplan-Meier plot showing the percentage of mice withmicrovascular occlusion as a function of time in FITC-Dextran inducedthrombotic microangiopathy in mice treated with increasing doses ofhuman MASP-2 inhibitory antibody (mAbH6 at 2 mg/kg, 10 mg/kg or 20mg/kg) or an isotype control antibody. As shown in FIG. 51, completemicrovascular occlusion was delayed in the mAbH6 treated groups ascompared to the control mice.FIG. 52 graphically illustrates the median time to microvascularocclusion as a function of mAbH6 dose (*p<0.05 compared to control). Asshown in FIG. 52, the median time to complete microvascular occlusionincreased from 23.3 minutes in the control group to 38.6 minutes in the2 mg/kg mAbH6 treated group (p<0.05). Doses of 10 mg/kg or 20 mg/kg ofmAbH6 performed similarly (median time for complete microvascularocclusion was 40.3 and 38 minutes, respectively) to the 2 mg/kg mAbH6treated group. The underlying experimental data and statistical analysisare provided in TABLES 16 and 17.The time to complete vessel occlusion in individual mice recorded basedon primary evaluation of the videographic recording is detailed below inTABLE 16.

TABLE 16 Time to Complete Occlusion After Light Dye-Induced InjuryControl Treatment mAbH6 Treatment Control 2 mg/kg 10 mg/kg 20 mg/kg Timeto 37.50 42.3 30.92 38.00 Occlusion 29.07 21.91 17.53 28.00 (minutes)27.12 24.4 51.38 40.58 19.38 31.38 36.88 33.00 19.55 61.17* 26.83 39.1018.00 61.55* 40.28 32.03 16.50 55.83 38.53 23.33 71.93* 21.75* 14.8398.22* 32.25* 30*   33.17* 61.8* *vessels did not completely occludeduring the indicated observation period.The statistical analysis comparing time to complete occlusion betweencontrol and mAbH6 treated animals is shown below in TABLE 17.

TABLE 17 Time to Complete Microvascular Occlusion: data from FITC Dexdose response study mAbH6 mAbH6 mAbH6 Statistic Control (2 mg/kg) (10mg/kg) (20 mg/kg) Number of events/number of 9/11 4/6 7/9 7/10 animals(%) (81.8%) (66.7%) (77.8%) (70.0%) Median time (minutes) (95% CI) 23.336.8 40.3 38.0 (16.5, 37.5) (21.9, NE) (17.5, NE) (28.0, 40.6) Wilcoxonp-value* 0.0456 0.0285 0.0260 Event = Time to occlusion observed Median(minutes) and its 95% CI were based on Kaplan-Meier estimate NE = notestimable *p-values were adjusted by Dunnett-Hsu multiple comparison

Summary

As summarized in TABLE 18, the initiation of thrombus formation wasdelayed in the mAbH6 treated mice in a dose-dependent manner relative tothe control-treated mice (median time to onset 10.4 to 17.7 minutes vs6.8 minutes). The median time to complete occlusion was significantlydelayed in all mAbH6-treated groups relative to the control-treatedgroups (Table 18).

TABLE 18 Median Time to Onset of Thrombus Formation and CompleteOcclusion mAbH6 mAbH6 mAbH6 Control (2 mg/kg) (10 mg/kg) (20 mg/kg)Median# time to 6.8 10.4 11.7 17.7* onset of thrombus formation(minutes) Median# time to 23.3 36.8* 40.3* 38.0* complete microvascularocclusion (minutes) #Median values are based on Kaplan-Meier estimate *p< 0.05 compared to control (Wilcoson adjusted by Dunnett-Hsu formultiple comparisons)

These results demonstrate that mAbH6, a human monoclonal antibody thatbinds to MASP-2 and blocks the lectin pathway of the complement system,reduced microvascular thrombosis in a dose-dependent manner in anexperimental mouse model of TMA. Therefore, it is expected thatadministration of a MASP-2 inhibitory agent, such as a MASP-2 inhibitoryantibody, will be an effective therapy in patients suffering from HUS,aHUS, TTP, or other microangiopathic disorders such as other TMAsincluding catastrophic antiphospholipid syndrome (CAPS), systemic Degosdisease, and TMAs secondary to cancer, cancer chemotherapy andtransplantation and provide protection from microvascular coagulationand thrombosis.

Example 40

This Example describes the identification, using phage display, of fullyhuman scFv antibodies that bind to MASP-2 and inhibit lectin-mediatedcomplement activation while leaving the classical (C1q-dependent)pathway and the alternative pathway components of the immune systemintact.

Overview:

Fully human, high-affinity MASP-2 antibodies were identified byscreening a phage display library. The variable light and heavy chainfragments of the antibodies were isolated in both a scFv format and in afull-length IgG format. The human MASP-2 antibodies are useful forinhibiting cellular injury associated with lectin pathway-mediatedalternative complement pathway activation while leaving the classical(Clq-dependent) pathway component of the immune system intact. In someembodiments, the subject MASP-2 inhibitory antibodies have the followingcharacteristics: (a) high affinity for human MASP-2 (e.g., a K_(D) of 10nM or less), and (b) inhibit MASP-2-dependent complement activity in 90%human serum with an IC₅₀ of 30 nM or less.

Methods:

Expression of Full-Length Catalytically Inactive MASP-2:

The full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4), encodingthe human MASP-2 polypeptide with leader sequence (SEQ ID NO:5) wassubcloned into the mammalian expression vector pCI-Neo (Promega), whichdrives eukaryotic expression under the control of the CMVenhancer/promoter region (described in Kaufman R. J. et al., NucleicAcids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology,185:537-66 (1991)). In order to generate catalytically inactive humanMASP-2A protein, site-directed mutagenesis was carried out as describedin US2007/0172483, hereby incorporated herein by reference. The PCRproducts were purified after agarose gel electrophoresis and bandpreparation and single adenosine overlaps were generated using astandard tailing procedure. The adenosine-tailed MASP-2A was then clonedinto the pGEM-T easy vector and transformed into E. coli. The humanMASP-2A was further subcloned into either of the mammalian expressionvectors pED or pCI-Neo.

The MASP-2A expression construct described above was transfected intoDXB1 cells using the standard calcium phosphate transfection procedure(Maniatis et al., 1989). MASP-2A was produced in serum-free medium toensure that preparations were not contaminated with other serumproteins. Media was harvested from confluent cells every second day(four times in total). The level of recombinant MASP-2A averagedapproximately 1.5 mg/liter of culture medium. The MASP-2A (Ser-Alamutant described above) was purified by affinity chromatography onMBP-A-agarose columns

MASP-2A ELISA on ScFv Candidate Clones Identified by Panning/scFvConversion and Filter Screening

A phage display library of human immunoglobulin light- and heavy-chainvariable region sequences was subjected to antigen panning followed byautomated antibody screening and selection to identify high-affinityscFv antibodies to human MASP-2 protein. Three rounds of panning thescFv phage library against HIS-tagged or biotin-tagged MASP-2A werecarried out. The third round of panning was eluted first with MBL andthen with TEA (alkaline). To monitor the specific enrichment of phagesdisplaying scFv fragments against the target MASP-2A, a polyclonal phageELISA against immobilized MASP-2A was carried out. The scFv genes frompanning round 3 were cloned into a pHOG expression vector and run in asmall-scale filter screening to look for specific clones againstMASP-2A.

Bacterial colonies containing plasmids encoding scFv fragments from thethird round of panning were picked, gridded onto nitrocellulosemembranes and grown overnight on non-inducing medium to produce masterplates. A total of 18,000 colonies were picked and analyzed from thethird panning round, half from the competitive elution and half from thesubsequent TEA elution. Panning of the scFv phagemid library againstMASP-2A followed by scFv conversion and a filter screen yielded 137positive clones. 108/137 clones were positive in an ELISA assay forMASP-2 binding (data not shown), of which 45 clones were furtheranalyzed for the ability to block MASP-2 activity in normal human serum.

Assay to Measure Inhibition of Formation of Lectin Pathway C3 Convertase

A functional assay that measures inhibition of lectin pathway C3convertase formation was used to evaluate the “blocking activity” of theMASP-2 scFv candidate clones. MASP-2 serine protease activity isrequired in order to generate the two protein components (C4b, C2a) thatcomprise the lectin pathway C3 convertase. Therefore, a MASP-2 scFv thatinhibits MASP-2 functional activity (i.e., a blocking MASP-2 scFv), willinhibit de novo formation of lectin pathway C3 convertase. C3 containsan unusual and highly reactive thioester group as part of its structure.Upon cleavage of C3 by C3 convertase in this assay, the thioester groupon C3b can form a covalent bond with hydroxyl or amino groups onmacromolecules immobilized on the bottom of the plastic wells via esteror amide linkages, thus facilitating detection of C3b in the ELISAassay.

Yeast mannan is a known activator of the lectin pathway. In thefollowing method to measure formation of C3 convertase, plastic wellscoated with mannan were incubated with diluted human serum to activatethe lectin pathway. The wells were then washed and assayed for C3bimmobilized onto the wells using standard ELISA methods. The amount ofC3b generated in this assay is a direct reflection of the de novoformation of lectin pathway C3 convertase. MASP-2 scFv clones atselected concentrations were tested in this assay for their ability toinhibit C3 convertase formation and consequent C3b generation.

Methods:

The 45 candidate clones identified as described above were expressed,purified and diluted to the same stock concentration, which was againdiluted in Ca⁺⁺ and Mg⁺⁺ containing GVB buffer (4.0 mM barbital, 141 mMNaCl, 1.0 mM MgCl₂, 2.0 mM CaCl₂, 0.1% gelatin, pH 7.4) to assure thatall clones had the same amount of buffer. The scFv clones were eachtested in triplicate at the concentration of 2 μg/mL. The positivecontrol was OMS100 Fab2 and was tested at 0.4 μg/mL. C3c formation wasmonitored in the presence and absence of the scFv/IgG clones.

Mannan was diluted to a concentration of 20 μg/mL (1 μg/well) in 50 mMcarbonate buffer (15 mM Na₂CO₃+35 mM NaHCO₃+1.5 mM NaN₃), pH 9.5 andcoated on an ELISA plate overnight at 4° C. The next day, themannan-coated plates were washed 3 times with 200 μl PBS. 100 μl of 1%HSA blocking solution was then added to the wells and incubated for 1hour at room temperature. The plates were washed 3 times with 200 μlPBS, and stored on ice with 200 μl PBS until addition of the samples.

Normal human serum was diluted to 0.5% in CaMgGVB buffer, and scFvclones or the OMS100 Fab2 positive control were added in triplicates at0.01 μg/mL; 1 μg/mL (only OMS100 control) and 10 μg/mL to this bufferand preincubated 45 minutes on ice before addition to the blocked ELISAplate. The reaction was initiated by incubation for one hour at 37° C.and was stopped by transferring the plates to an ice bath. C3bdeposition was detected with a Rabbit α-Mouse C3c antibody followed byGoat α-Rabbit HRP. The negative control was buffer without antibody (noantibody=maximum C3b deposition), and the positive control was bufferwith EDTA (no C3b deposition). The background was determined by carryingout the same assay except that the wells were mannan-free. Thebackground signal against plates without mannan was subtracted from thesignals in the mannan-containing wells. A cut-off criterion was set athalf of the activity of an irrelevant scFv clone (VZV) and buffer alone.

Results:

Based on the cut-off criterion, a total of 13 clones were found to blockthe activity of MASP-2. All 13 clones producing >50% pathway suppressionwere selected and sequenced, yielding 10 unique clones. All ten cloneswere found to have the same light chain subclass, λ3, but threedifferent heavy chain subclasses: VH2, VH3 and VH6. In the functionalassay, five out of the ten candidate scFv clones gave IC₅₀ nM valuesless than the 25 nM target criteria using 0.5% human serum.

To identify antibodies with improved potency, the three mother scFvclones, identified as described above, were subjected to light-chainshuffling. This process involved the generation of a combinatoriallibrary consisting of the VH of each of the mother clones paired up witha library of naïve, human lambda light chains (VL) derived from sixhealthy donors. This library was then screened for scFv clones withimproved binding affinity and/or functionality.

TABLE 19 Comparison of functional potency in IC₅₀ (nM) of the leaddaughter clones and their respective mother clones (all in scFv format)1% human 90% human 90% human serum serum serum C3 assay C3 assay C4assay scFv clone (IC₅₀ nM) (IC₅₀ nM) (IC₅₀ nM) 17D20mc 38 nd nd17D20m_d3521N11 26 >1000 140 17N16mc 68 nd nd 17N16m_d17N9 48 15 230

Presented below are the heavy-chain variable region (V_(H)) sequencesfor the mother clones and daughter clones shown above in TABLE 19, andlisted below in TABLES 20A-F.

The Kabat CDRs (31-35 (H1), 50-65 (H2) and 95-102 (H3)) are bolded; andthe Chothia CDRs (26-32 (H1), 52-56 (H2) and 95-101 (H3)) areunderlined.

17D20_35VH-21N11VL heavy chain variable region (VH)(SEQ ID NO: 67, encoded by SEQ ID NO: 66)QVTLKESGPVLVKPTETLTLTCTVSGFSLSRG KMGVSWIR QPPGKALEWLA HIFSSDEKSYRTSLKSRLTISKDTSKNQ VVLTMTNMDPVDTAT YYCARIR RGGIDYWGQGTLVTVSSd17N9 heavy chain variable region (VH) (SEQ ID NO: 68)QVQLQQSGPGLVKPSQTLSLTCAISGDSVSST SAAWNWI RQSPSRGLEWLG RTYYRSKWYNDYAVSVKSRITINPDTS KNQFSLQLNSVTPEDT AVYYCAR DPFGVPFDIWGQGTMV TVSS

Heavy Chain Variable Region

TABLE 20A Heavy chain (aa 1-20) Heavy chain aa 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19 20 d3521N11 Q V T L K E S G P V L V K P T E T LT L (SEQ: 67) d178N9 Q V Q L Q Q S G P G L V K P S Q T L S L (SEQ: 68)

TABLE 20B Heavy chain (aa 21-40) Heavy chain CDR-H1 aa 21 22 23 24 25 2627 28 29 30 31 32 33 34 35 36 37 38 39 40 d3521N11 T C T V S G F S L S RG K M G V S W I R (SEQ: 67) d17N9 T C A I S G D S V S S T S A A W N W IR (SEQ: 68)

TABLE 20C Heavy chain (aa 41-60) Heavy chain CDR-H2 aa 41 42 43 44 45 4647 48 49 50 51 52 53 54 55 56 57 58 59 60 d3521N11 Q P P G K A L E W L AH I F S S D E K S (SEQ: 67) d17N9 Q S P S R G L E W L G R T Y Y R S K WY (SEQ: 68)

TABLE 20D Heavy chain (aa 61-80) Heavy chain CDR-H2 (cont'd) aa 61 62 6364 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 d3521N11 Y R T S L KS R L T I S K D T S K N Q V (SEQ: 67) d17N9 N D Y A V S V K S R I T I NP D T S K N (SEQ: 68)

TABLE 20E Heavy chain (aa 81-100) Heavy chain CDR-H3 aa 81 82 83 84 8586 87 88 89 90 91 92 93 94 95 96 97 98 99 100 d3521N11 V L T M T N M D PV D T A T Y Y C A R I (SEQ: 67) d17N9 Q F S L Q L N S V T P E D T A V YY C A (SEQ: 68)

TABLE 20F heavy chain (aa 101-118) Heavy chain CDR-H3 (cont'd) aa 101102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119120 d3521N11 R R G G I D Y W G Q G T L V T V S S (SEQ: 67) d17N9 R D P FG V P F D I W G Q G T M V T V S (SEQ: 68)

Presented below are the light-chain variable region (V_(L)) sequencesfor the mother clones and daughter clones listed below in TABLES 21A-F.

The Kabat CDRs (24-34 (L1); 50-56 (L2); and 89-97 (L3) are bolded; andthe Chothia CDRs (24-34 (L1); 50-56 (L2) and 89-97 (L3) are underlined.These regions are the same whether numbered by the Kabat or Chothiasystem.

17D20m_d3521N11 light chain variable region (VL)(SEQ ID NO: 70, encoded by SEQ ID NO: 69)QPVLTQPPSLSVSPGQTASITCSGEKLGDKYAYWYQQKPGQSPVLVMY Q DKQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ AWDSSTAV F GGGTKLTVL17N16m_d17N9 light chain variable region (VL) (SEQ ID NO: 71)SYELIQPPSVSVAPGQTATITCA GDNLGKKRVHW YQQRPGQAPVLVIY D DSDRPSGIPDRFSASNSGNTATLTITRGEAGDEADYYCQ VWDIATDH V VFGGGTKLTVLAAAGSEQKLISE

TABLE 21A Light chain (aa 1-20) Light chain aa 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19 20 d3521N11 Q P V L T Q P P S L S V S P G Q T AS I (SEQ: 67) d178N9 S Y E L I Q P P S V S V A P G Q T A T I (SEQ: 68)

TABLE 21B Light chain (aa 21-40) Light chain CDR-L1 aa 21 22 23 24 25 2627 28 29 30 31 32 33 34 35 36 37 38 39 40 d3521N11 T C S G E K L G D K YA Y W Y Q Q K P G (SEQ: 70) d17N9 T C A G D N L G K K R V H W Y Q Q R PG (SEQ: 71)

TABLE 21C Light chain (aa 41-60) Light chain CDR-L2 aa 41 12 43 44 45 4647 48 49 50 51 52 53 54 55 56 57 58 59 60 d3521N11 Q S P V L V M Y Q D KQ R P S G I P E R (SEQ: 70) d17N9 Q A P V L V I Y D D S D R P S G I P DR (SEQ: 71)

TABLE 21D Light chain (aa 61-80) Light chain CDR-L2 (cont'd) aa 61 62 6364 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 d3521N11 F S G S N SG N T A T L T I S G T Q A M (SEQ: 70) d17N9 F S A S N S G N T A T L T IT R G E A G (SEQ: 71)

TABLE 21E Light chain (aa 81-100) Light chain CDR-L3 aa 81 82 83 84 8586 87 88 89 90 91 92 93 94 95 96 97 98 99 100 d3521N11 D E A D Y Y C Q AW D S S T A V F G G G (SEQ: 70) d17N9 D E A D Y Y C Q V W D I A T D H VV F G (SEQ: 71)

TABLE 21F Light chain (aa 101-120) Light chain CDR-L3 (cont'd) aa 101102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119120 d3521N11 T K L T V L A A A G S E Q K L I S E E D (SEQ: 70) d17N9 G GT K L T V L A A A G S E Q K L I S E (SEQ: 71)

The MASP-2 antibodies OMS100 and MoAb_d3521N11VL, (comprising a heavychain variable region set forth as SEQ ID NO:67 and a light chainvariable region set forth as SEQ ID NO:70, also referred to as “OMS646”and “mAbH6”), which have both been demonstrated to bind to human MASP-2with high affinity and have the ability to block functional complementactivity, were analyzed with regard to epitope binding by dot blotanalysis. The results show that OMS646 and OMS100 antibodies are highlyspecific for MASP-2 and do not bind to MASP-1/3. Neither antibody boundto MAp19 nor to MASP-2 fragments that did not contain the CCP1 domain ofMASP-2, leading to the conclusion that the binding sites encompass CCP1.

The MASP-2 antibody OMS646 was determined to avidly bind to recombinantMASP-2 (Kd 60-250 pM) with >5000 fold selectivity when compared to C1s,C1r or MASP-1 (see TABLE 22 below):

TABLE 22 Affinity and Specificity of OM5646 MASP-2 antibody-MASP-2interaction as assessed by solid phase ELISA studies Antigen K_(D) (pM)MASP-1 >500,000 MASP-2 62 ± 23* MASP-3 >500,000 Purified humanC1r >500,000 Purified human C1s ~500,000 *Mean ± SD; n = 12

OMS646 Specifically Blocks Lectin-Deendent Activation of TerminalComplement Components

Methods:

The effect of OMS646 on membrane attack complex (MAC) deposition wasanalyzed using pathway-specific conditions for the lectin pathway, theclassical pathway and the alternative pathway. For this purpose, theWieslab Comp300 complement screening kit (Wieslab, Lund, Sweden) wasused following the manufacturer's instructions.

Results:

FIG. 53A graphically illustrates the level of MAC deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) under lectinpathway-specific assay conditions. FIG. 53B graphically illustrates thelevel of MAC deposition in the presence or absence of anti-MASP-2antibody (OMS646) under classical pathway-specific assay conditions.FIG. 53C graphically illustrates the level of MAC deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) under alternativepathway-specific assay conditions.

As shown in FIG. 53A, OMS646 blocks lectin pathway-mediated activationof MAC deposition with an IC₅₀ value of approximately InM. However,OMS646 had no effect on MAC deposition generated from classicalpathway-mediated activation (FIG. 53B) or from alternativepathway-mediated activation (FIG. 53C).

Pharmacokinetics and Pharmacodynamics of 0MS646 Following Intravenous(IV) or Subcutaneous (SC) Administration to Mice

The pharmacokinetics (PK) and pharmacodynamics (PD) of OMS646 wereevaluated in a 28 day single dose PK/PD study in mice. The study testeddose levels of 5 mg/kg and 15 mg/kg of OMS646 administeredsubcutaneously (SC), as well as a dose level of 5 mg/kg OMS646administered intravenously (IV).

With regard to the PK profile of OMS646, FIG. 54 graphically illustratesthe OMS646 concentration (mean of n=3 animals/groups) as a function oftime after administration of OMS646 at the indicated dose. As shown inFIG. 54, at 5 mg/kg SC, OMS646 reached the maximal plasma concentrationof 5-6 ug/mL approximately 1-2 days after dosing. The bioavailability ofOMS646 at 5 mg/kg SC was approximately 60%. As further shown in FIG. 54,at 15 mg/kg SC, OMS646 reached a maximal plasma concentration of 10-12ug/mL approximately 1 to 2 days after dosing. For all groups, the OMS646was cleared slowly from systemic circulation with a terminal half-lifeof approximately 8-10 days. The profile of OMS646 is typical for humanantibodies in mice. The PD activity of OMS646 is graphically illustratedin FIGS. 55A and 55B.

FIGS. 55A and 55B show the PD response (drop in systemic lectin pathwayactivity) for each mouse in the 5 mg/kg IV (FIG. 55A) and 5 mg/kg SC(FIG. 55B) groups. The dashed line indicates the baseline of the assay(maximal inhibition; naïve mouse serum spiked in vitro with excessOMS646 prior to assay). As shown in FIG. 55A, following IVadministration of 5 mg/kg of OMS646, systemic lectin pathway activityimmediately dropped to near undetectable levels, and lectin pathwayactivity showed only a modest recovery over the 28 day observationperiod. As shown in FIG. 55B, in mice dosed with 5 mg/kg of OMS646 SC,time-dependent inhibition of lectin pathway activity was observed.Lectin pathway activity dropped to near-undetectable levels within 24hours of drug administration and remained at low levels for at least 7days. Lectin pathway activity gradually increased with time, but did notrevert to pre-dose levels within the 28 day observation period. Thelectin pathway activity versus time profile observed afteradministration of 15 mg/kg SC was similar to the 5 mg/kg SC dose (datanot shown), indicating saturation of the PD endpoint. The data furtherindicated that weekly doses of 5 mg/kg of OMS646, administered either IVor SC, is sufficient to achieve continuous suppression of systemiclectin pathway activity in mice.

Example 41

This Example demonstrates that a MASP-2 inhibitory antibody (OMS646)inhibits aHUS serum-induced complement C5b-9 deposition on the surfaceof activated human microvascular endothelial cells (HMEC-1) afterexposure to serum from patients with atypical hemolytic uremic syndrome(aHUS) obtained during the acute phase and the remission phase of thedisease.

Background/Rationale:

The following study was carried out to analyze aHUS serum-inducedcomplement C5b-9 deposition on the surface of activated HMEC-1 cellsafter exposure to aHUS patient serum obtained (1) during the acute phaseand (2) during the remission phase of the disease in the presence orabsence of OMS646, a MASP-2 antibody that specifically binds to MASP-2and inhibits lectin pathway activation.

Methods:

Patients:

Four patients with aHUS, studied both during the acute phase of thedisease and in remission, were selected for this study among thoseincluded in the International Registry of HUS/TTP and genotyped by theLaboratory of Immunology and Genetics of Transplantation and RareDiseases of the Mario Negri Institute. One aHUS patient had aheterozygous p.R1210C complement factor H (CFH) mutation and one hadanti-CFH autoantibodies, while no mutation or antibodies to CFH werefound in the other two aHUS patients.

Tables 23 and 24 summarize the results of screening for complement genemutations and anti-CFH autoantibodies in the four aHUS patients analyzedin this study along with clinical and biochemical data measured eitherduring the acute phase or at remission.

TABLE 23 Clinical Parameters of the four aHUS patients in this studyPlatelets LDH s-Creatinine Case Mutation or anti- Disease (150-400*(266-500 Hemoglobin (0.55-1.25 No. CFH Ab phase 10³/ul) IU/l) (14-18g/dl) mg/dl) #1 no mutations, no acute 31,000 1396 12.9 2.37 anti-CFH Abremission 267,000 n.a. 11.5 3.76 #2 CFH-R1210C acute 46,000 1962 7 5.7remission 268,000 440 13.4 7.24 #3 anti-CFH Ab acute 40,000 3362 9.51.77 remission 271,000 338 8.8 0.84 #4 no mutations, no acute 83,0001219 7.8 6.8 anti-CFH Ab remission 222,000 495 12.2 13 Note: n.a. = notavailable

TABLE 24 Complement Parameters of the four aHUS patients in this studyCase Mutation of anti- Disease Serum C3 Plasma SC5b-9 No. CFH Ab phase(83-180 mg/dl) (127-400 ng/ml) #1 no mutations, no acute 51 69 anti-CFHAb remission n.a. 117 #2 CFH-R1210C acute 79 421 remission 119 233 #3anti-CFH Ab acute 58 653 remission 149 591 #4 no mutations, no acute 108n.a. anti-CFH Ab remission n.a. n.a.

Experimental Methods:

Cells from a human microvascular endothelial cell line (HMEC-1) ofdermal origin were plated on glass slides and used when confluent.Confluent HMEC-1 cells were activated with 10 M ADP (adenosinediphosphate) for 10 minutes and then incubated for four hours with serumfrom the four aHUS patients described above in Tables 23 and 24collected either during the acute phase of the disease, or from the sameaHUS patients at remission, or from 4 healthy control subjects. Theserum was diluted 1:2 with test medium (HBSS with 0.5% BSA) in thepresence or in the absence of a MASP-2 inhibitory antibody, OMS646 (100μg/mL), generated as described above in Example 40, or in the presenceof soluble complement receptor 1 (sCRI) (150 μg/mL), as a positivecontrol of complement inhibition. At the end of the incubation step, theHMEC-1 cells were treated with rabbit anti-human complement C5b-9followed by FITC-conjugated secondary antibody. In each experiment,serum from one healthy control was tested in parallel with aHUS patientserum (acute phase and remission). A confocal inverted laser microscopewas used for acquisition of the fluorescent staining on the endothelialcell surface. Fifteen fields per sample were acquired and the areaoccupied by the fluorescent staining was evaluated by automatic edgedetection using built-in specific functions of the software Image J andexpressed as pixel² per field analyzed. The fields showing the lowestand the highest values were excluded from calculation.

For the statistical analysis (one-way ANOVA followed by Tukey's test formultiple comparisons) results in pixel² of the 13 fields considered ineach experimental condition for each patient and control were used.

Results:

The results of the complement deposition analysis with the sera from thefour aHUS patients are summarized below in Table 25A, and the resultswith the sera from the four healthy subjects are summarized below inTable 25B.

TABLE 25A Effect of complement inhibitors on aHUS serum-induced C5b-9deposition on ADP-activated HMEC-1 cells aHUS aHUS acute phase aHUSremission phase Patient # untreated +sCR1 +OMS646 untreated +sCR1+OMS646 Patient #1 5076 ± 562° 551 ± 80* 3312 ± 422** 4507 ± 533° 598 ±101§ 1650 ± 223§ (no mutation, no anti-CFH ab) Patient #2 5103 ± 648°497 ± 67* 2435 ± 394* 3705 ± 570° 420 ± 65§ 2151 ± 250§§§ (CFH-R1210C)Patient #3 3322 ± 421° 353 ± 64* 2582 ± 479 6790 ± 901° 660 ± 83§ 2077 ±353§ (anti-CFH ab) Patient #4 4267 ± 488° 205 ± 34* 2369 ± 265** 5032 ±594° 182 ± 29§ 3290 ± 552§§ (no mutations, no anti-CFH ab)

TABLE 25B Effect of complement inhibitors on sera from four healthycontrol subjects (not suffering from aHUS) on C5b-9 deposition onADP-activated HMEC-1 cells Healthy Control Subject # Untreated +sCR1+OMS646 Control Subject #1 481 ± 66 375 ± 43 213 ± 57 (assayed inparallel with aHUS subject #1) Control Subject #2 651 ± 61 240 ± 33 490± 69 (assayed in parallel with aHUS subject #2) Control Subject #3 602 ±83 234 ± 35  717 ± 109 (assayed in parallel with aHUS subject #3)Control Subject #4 370 ± 53 144 ± 20 313 ± 36 (assayed in parallel withaHUS subject #4)For Tables 25A and 25B: Data are mean+SE. ^(o)P<0.001 vs control;*P<0.001, **P<0.01 vs aHUS acute phase untreated; § P<0.001, § § P<0.01,§ § § P<0.05 vs aHUS remission phase untreated.

FIG. 56 graphically illustrates the inhibitory effect of MASP-2 antibody(OMS646) and sCRI on aHUS serum-induced C5b-9 deposition onADP-activated HMEC-1 cells. In FIG. 56, the data are mean±SE.^(o)P<0.0001 vs control; *P<0.0001 vs aHUS acute phase untreated;{circumflex over ( )}P<0.0001 vs aHUS acute phase+sCRI; § P<0.0001 vsaHUS remission phase untreated and # P<0.0001 vs aHUS remissionphase+sCR1.

As shown in Table 25A, 25B and FIG. 56, ADP-stimulated HMEC-1 cellsexposed to serum from aHUS patients (collected either in the acute phaseor in remission) for four hours in static conditions showed an intensedeposition of C5b-9 on cell surface as detected by confocal microscopy.By measuring the area covered by C5b-9, a significantly higher amount ofC5b-9 deposition was observed on cells exposed to serum from aHUSpatients than on cells exposed to serum from healthy control subjects,irrespective of whether aHUS serum was collected in the acute phase orduring remission. No difference in serum-induced endothelial C5b-9deposits was observed between acute phase and remission.

As further shown in Table 25A, 25B and FIG. 56, addition of the MASP-2antibody OMS646 to aHUS serum (either obtained from patients duringacute phase or in remission) led to a significant reduction of C5b-9deposition on endothelial cell surface as compared to untreated aHUSserum. However, the inhibitory effect of OMS646 on C5b-9 deposition wasless profound than the effect exerted by the complement pan-inhibitorsCR1. Indeed, a statistically significant difference was observedbetween aHUS serum-induced C5b-9 deposits in the presence of OMS646 vs.sCRI (FIG. 56 and Tables 25A and 25B).

When calculated as a mean of the four aHUS patients, the percentages ofreduction of C5b-9 deposits (as compared with C5b-9 deposits induced bythe untreated serum from the same patients taken as 100%) observed inthe presence of the complement inhibitors were as follows:

Acute Phase:

-   -   sCRI (150 μg/ml): 91% reduction in C5b-9 deposits    -   OMS646 (100 μg/ml): 40% reduction in C5b-9 deposits

Remission Phase:

-   -   sCRI (150 μg/ml): 91% reduction in C5b-9 deposits    -   OMS646 (100 μg/ml): 54% reduction in C5b-9 deposits

Conclusion:

The results described in this Example demonstrate that the lectinpathway of complement is stimulated by activated microvascularendothelial cells and that this stimulation is a significant driver forthe exaggerated complement activation response characteristic of aHUS.It is also demonstrated that this stimulation of the lectin pathway andresulting exaggerated complement activation response occurs both duringthe acute phase and in clinical remission of aHUS. Moreover, thisfinding does not appear to be limited to any particular complementdefect associated with aHUS. As further demonstrated in this Example,selective inhibition of the lectin pathway with a MASP-2 inhibitoryantibody such as OMS646 reduces complement deposition in aHUS patientswith diverse etiologies.

Example 42

This Example demonstrates that a MASP-2 inhibitory antibody (OMS646)inhibits aHUS serum-induced platelet aggregation and thrombus formationon the surface of activated human microvascular endothelial cells(HMEC-1) after exposure to aHUS patient serum obtained during (1) theacute phase and (2) the remission phase of aHUS.

Methods:

Patients:

Three patients (patients #1, #2 and #4 as described in Tables 23, 24,25A and 25B in Example 41) with aHUS (one patient had a heterozygousp.R1210C CFH mutation, while no mutation or anti-CFH antibodies werefound in the other two patients) were studied both during the acutephase of the disease and in remission. The patients were selected forthis study among those included in the International Registry of HUS/TTPand genotyped by the Laboratory of Immunology and Genetics ofTransplantation and Rare Diseases of the Mario Negri Institute. Fivehealthy subjects were also selected as blood donors for perfusionexperiments.

Methods:

Confluent HMEC-1 cells were activated with 10 μM ADP for 10 minutes andthen were incubated for three hours with sera from three aHUS patients(patients #1, 2 and 4 described in Example 41) collected during theacute phase of the disease or from the same patients at remission, orwith control sera from healthy subjects. The serum was diluted 1:2 withtest medium (HBSS with 0.5% BSA), in the presence or in the absence of aMASP-2 inhibitory antibody, OMS646 (100 μg/mL), generated as describedin Example 40; or with sCRI (150 μg/mL), as a positive control ofcomplement inhibition. For patients #1 and #2 additional wells wereincubated with sera (from acute phase and remission) diluted 1:2 withtest medium containing 100 μg/mL of irrelevant isotype control antibodyor with 20 μg/mL of OMS646 (for the latter, case #1 was tested only inremission and case #2 both during the acute phase and at remission).

At the end of the incubation step, HMEC-1 cells were perfused in a flowchamber with heparinized whole blood (10 UI/mL) obtained from healthysubjects (containing the fluorescent dye mepacrine that labelsplatelets) at the shear stress encountered in the microcirculation (60dynes/cm², three minutes). After three minutes of perfusion, theendothelial-cell monolayers were fixed in acetone. Fifteen images persample of platelet thrombi on the endothelial cell surface were acquiredby confocal inverted laser microscope, and areas occupied by thrombiwere evaluated using Image J software. The fields showing the loweestand the highest values were excluded from calculation.

For statistical analysis (one-way ANOVA followed by Tukey's test formultiple comparisons), results in pixel² of the 13 fields considered ineach experimental condition for each patient and control were used.

Results:

The results of the thrombus formation experiments with the sera from thethree aHUS patients are summarized below in Table 26A, and the resultswith the sera from the five healthy subjects are summarized below inTable 26B.

TABLE 26A Effect of complement inhibitors on aHUS serum-induced thrombusformation (pixel² ± SE) on ADP-activated HMEC-1 Cells aHUS Case #1 aHUSaHUS Case #4 thrombus Case #2 thrombus formation thrombus formation(pixel² ± SE) formation (pixel² ± SE) (no (pixel² ± SE) (no mutations,Experimental Disease mutation, no (CFH- no anti- conditions phaseanti-CFH ab) R1210C) CFH ab) untreated acute 5499 ± 600 22320 ± 1273°10291 ± 1362° remission 6468 ± 1012° 3387 ± 443° 17676 ± 1106° +sCR1acute 4311 ± 676 5539 ± 578* 5336 ± (150 μg/mL) remission 573 ± 316§ 977± 102§ 1214*** 2544 ± 498§ +OMS646 acute not determined 6974 ± 556* notdetermined (20 μg/mL) remission 832 ± 150§ 1224 ± 252§  not determined+OMS646 acute 3705 ± 777 9913 ± 984* 2836 ± 509* (100 μg/mL) remission3321 ± 945§§§ 733 ± 102§ 1700 ± 321§ +irrelevant acute 5995 ± 725 18655± 1699  not determined isotype remission 10885 ± 1380 2711 ± 371  notdetermined control antibody (100 μg/mL)

TABLE 26B Effect of complement inhibitors on sera from five healthycontrol subjects (not suffering from aHUS) in the thrombus formation(pixel² ± SE) assay on ADP-activated HMEC-1 Cells Control #1 Control #2Control #3 Control #4 Control #5 thrombus thrombus thrombus thrombusthrombus Experimental formation formation formation formation formationconditions (pixel² ± SE) (pixel² ± SE) (pixel² ± SE) (pixel² ± SE)(pixel² ± SE) untreated 2880 ± 510 1046 ± 172 1144 ± 193 735 ± 124 2811± 609  +sCR1 5192 ± 637 1527 ± 153 1198 ± 138 2239 ± 243  2384 ± 410 (150 μg/mL) +OMS646 7637 ± 888 1036 ± 175  731 ± 203 2000 ± 356  7177 ±1477 (100 μg/mL) +irrelevant 6325 ± 697 1024 ± 235 399 ± 82 45269 notisotype control determined antibody (100 μg/mL Assayed in #1 (acute #1(remission #2 (acute #2 (remission #5 (acute and parallel with phaseserum) phase serum) phase serum) phase serum) remission serum from phaseserum) aHUS subjectFor Tables 26A and 26B: Data are mean+SE. ^(o)P<0.001 vs control;*P<0.001, ***P<0.05 vs aHUS acute phase untreated; § P<0.001, § § §P<0.05 vs aHUS remission phase untreated.

FIG. 57 graphically illustrates the effect of MASP-2 antibody (OMS646)and sCRI on aHUS serum-induced thrombus formation on ADP-activatedHMEC-1 cells. In FIG. 57, the data shown are mean+SE. ^(o)P<0.0001,^(oo)P<0.01 vs control; *P<0.0001, **P<0.01 vs aHUS acute phaseuntreated; § P<0.0001 vs aHUS remission phase untreated.

As shown in Table 26A and FIG. 57, a marked increase in the area coveredby thrombi was observed on HMEC-1 cells treated with aHUS serum,collected either during acute phase or at remission, in comparison tocells exposed to serum from healthy control subjects (Table 26B and FIG.57). As shown in FIG. 57 and Table 26A, OMS646 (at both 100 μg/ml and 20μg/ml) showed a partial inhibition of thrombus formation on cellspre-exposed to aHUS serum taken during the acute phase. Theanti-thrombogenic effect was comparable between the two different dosesof OMS646 and was not different from the effect of sCRI (FIG. 57 andTable 26A). Addition of the irrelevant isotype control antibody had noinhibitory effect on aHUS-serum-induced thrombus formation.

As further shown in FIG. 57 and Table 26A, the inhibitory effect ofOMS646 was even more evident on aHUS serum collected during remissionphase. Indeed, the addition of OMS646, at both 100 μg/ml and 20 μg/mldoses, to aHUS patient serum collected at remission resulted in a nearlycomplete inhibition of thrombus formation, similar to that observed withthe addition of sCR1. The irrelevant isotype control antibody showed nosignificant inhibitory effect.

When calculated as a mean of the three aHUS patients, the percentages ofreduction of the HMEC-1 surface covered by thrombi deposits (as comparedwith thrombus area induced by the untreated sera from the same patientstaken as 100%) recorded with the complement inhibitors were as follows:

Acute Phase:

-   -   sCRI (150 μg/ml): 60% reduction    -   OMS646 (100 μg/ml): 57% reduction    -   OMS646 (20 μg/ml): 45% reduction

Remission Phase:

-   -   sCR1 (150 μg/ml): 85% reduction    -   OMS646 (100 μg/ml): 79% reduction    -   OMS646 (20 μg/ml): 89% reduction

Discussion of Results:

The results in this Example demonstrate that a MASP-2 inhibitoryantibody, such as OMS646 (generated as described in Example 40), has astrong inhibitory effect on aHUS serum-induced thrombus formation onHMEC-1 cells. Surprisingly, the inhibitory effect of OMS646 on thrombusformation was greater than its effect on C5b-9 deposits induced onHMEC-1 (as described in Example 41). It is also surprising that theaddition of OMS646, at both 100 μg/ml and 20 μg/ml doses, to aHUSpatient serum collected at remission resulted in nearly a completeinhibition of thrombus formation. Another surprising finding is theobservation that OMS646, in both the acute phase and in remission, wasas effective as the positive control sCR1, which is a broad and almostcomplete inhibitor of the complement system (Weisman H. et al., Science249:146-151, 1990; Lazar H. et al., Circulation 100:1438-1442, 1999).

It is noted that the control serum from healthy subjects also induced amodest thrombus formation on HMEC-1 cells. We did not observe aconsistent inhibitory effect on control serum induced thrombus formationwith either OMS646 or with sCR1. While not wishing to be bound by anyparticular theory, it is believed that the control-induced thrombi donot depend upon complement, as supported by very low C5b-9 depositsobserved on HMEC-1 incubated with control serum (see Example 41).

Conclusion:

In conclusion, the observed anti-thrombotic effect of a MASP-2inhibitory antibody, such as OMS646, appears substantially greater thanone would have expected based on the inhibitory effect of OMS646 onC5b-9 deposition observed in this experimental system (as described inExample 41 and shown in FIG. 56). For example, as described in Gastoldiet al., Immunobiology 217:1129-1222 Abstract 48 (2012) entitled“C5a/C5aR interaction mediates complement activation and thrombosis onendothelial cells in atypical hemolytic uremic syndrome (aHUS),” it wasdetermined that addition of a C5 antibody inhibiting C5b-9 deposits (60%reduction) limited thrombus formation on HMEC-1 to a comparable extent(60% reduction). In contrast, the MASP-2 inhibitory antibody (OMS646 at100 μg/mL) inhibited C5b-9 deposits with mean values of (acute phase=40%reduction; remission phase=54% reduction); and inhibited thrombusformation at a substantially higher percent (acute phase=57% reduction;remission phase=79% reduction). In comparison, OMS646 inhibitedcomplement deposition at a lower percentage than did the positivecontrol complement inhibitor (sCRI at 150 μg/mL, acute phase inhibitionof C5b-9 deposition=91% reduction; remission phase=91% reduction) yetwas equally effective as the sCRI positive control in inhibitingthrombus formation (sCRI at 150 μg/mL, acute phase=60% reduction;remission phase=85% reduction). These results demonstrate that a MASP-2inhibitory antibody (e.g., OMS646) is surprisingly effective atinhibiting thrombus formation in serum obtained from aHUS subjects bothin the acute phase and remission phase. In accordance with theforegoing, in one embodiment, the invention provides a method ofinhibiting thrombus formation in a subject suffering from, or at riskfor developing, a thrombotic microangiopathy (TMA) comprisingadministering to the subject a composition comprising an amount of aMASP-2 inhibitory antibody effective to inhibit MASP-2-dependentcomplement activation. In one embodiment, the TMA is selected from thegroup consisting of hemolytic uremic syndrome (HUS), thromboticthrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome(aHUS). In one embodiment, the TMA is aHUS. In one embodiment, thecomposition is administered to an aHUS patient during the acute phase ofthe disease. In one embodiment, the composition is administered to anaHUS patient during the remission phase (i.e., in a subject that hasrecovered or partially recovered from an episode of acute phase aHUS,such remission evidenced, for example, by increased platelet countand/or reduced serum LDH concentrations, for example as described inLoirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, herebyincorporated herein by reference).

In one embodiment, the MASP-2 inhibitory antibody exhibits at least oneor more of the following characteristics: said antibody binds humanMASP-2 with a K_(D) of 10 nM or less, said antibody binds an epitope inthe CCP1 domain of MASP-2, said antibody inhibits C3b deposition in anin vitro assay in 1% human serum at an IC₅₀ of 10 nM or less, saidantibody inhibits C3b deposition in 90% human serum with an IC₅₀ of 30nM or less, wherein the antibody is an antibody fragment selected fromthe group consisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)₂, wherein theantibody is a single-chain molecule, wherein said antibody is an IgG2molecule, wherein said antibody is an IgG1 molecule, wherein saidantibody is an IgG4 molecule, wherein the IgG4 molecule comprises aS228P mutation, and/or wherein the antibody does not substantiallyinhibit the classical pathway. In one embodiment, the antibody binds toMASP-2 and selectively inhibits the lectin pathway and does notsubstantially inhibit the alternative pathway. In one embodiment, theantibody binds to MASP-2 and selectively inhibits the lectin pathway anddoes not substantially inhibit the classical pathway or the alternativepathway (i.e., inhibits the lectin pathway while leaving the classicaland alternative complement pathways intact).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from a subject suffering from a TMA such as aHUS(acute or remission phase), by at least 30%, such as at least 40%, suchas at least 50%, such as at least 60%, such as at least 70%, such as atleast 80% such as at least 85%, such as at least 90%, such as at least95% up to 99%, as compared to untreated serum. In some embodiments, theMASP-2 inhibitory antibody inhibits thrombus formation in serum from asubject suffering from aHUS at a level of at least 20 percent orgreater, (such as at least 30%, at least 40%, at least 50%) more thanthe inhibitory effect on C5b-9 deposition in serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombusformation in serum from an aHUS patient in remission phase by at least30%, such as at least 40%, such as at least 50%, such as at least 60%,such as at least 70%, such as at least 80% such as at least 85%, such asat least 90%, such as at least 95% up to 99%, as compared to untreatedserum. In some embodiments, the MASP-2 inhibitory antibody inhibitsthrombus formation in serum in an aHUS patient in remission phase at alevel of at least 20 percent or greater, (such as at least 30%, at least40%, at least 50%) more than the inhibitory effect on C5b-9 depositionin serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to thesubject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibitingthrombus formation in a subject suffering from a TMA comprisingadministering to the subject a composition comprising an amount of aMASP-2 inhibitory antibody, or antigen binding fragment thereof,comprising (I) (a) a heavy-chain variable region comprising: i) aheavy-chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acidsequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and b) alight-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70, or (II) a variant thereofcomprising a heavy-chain variable region with at least 90% identity toSEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to SEQ ID NO:67) and a light-chain variable region with atleast 90% identity (e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to SEQ ID NO:70.

In one embodiment, the TMA is selected from the group consisting ofatypical hemolytic uremic syndrome (aHUS) (either acute or remissionphase), HUS and TTP. In one embodiment, the subject is in acute phase ofaHUS. In one embodiment, the subject is in remission phase of aHUS.

In some embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a heavy-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:67. Insome embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a light-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, that specifically recognizes at least part of anepitope on human MASP-2 recognized by reference antibody OMS646comprising a heavy-chain variable region as set forth in SEQ ID NO:67and a light-chain variable region as set forth in SEQ ID NO:70.Competition between binding members may be assayed easily in vitro, forexample using ELISA and/or by tagging a specific reporter molecule toone binding member which can be detected in the presence of otheruntagged binding member(s), to enable identification of specific bindingmembers which bind the same epitope or an overlapping epitope. Thus,there is presently provided a specific antibody or antigen-bindingfragment thereof, comprising a human antibody antigen-binding site,which competes with reference antibody OMS646 for binding to humanMASP-2.

Example 43

This Example demonstrates that a human MASP-2 inhibitory antibody(OMS646) is able to inhibit TMA patient plasma-mediated induction ofapoptosis in primary human microvascular endothelial cells (MVECs) ofdermal origin.

Background/Rationale:

The pathophysiology of TMA is known to involve an endothelial cellinjury induced by various factors that is followed by occlusions ofsmall vessels (e.g., small arterioles and capillaries) by platelet plugsand/or fibrin thrombi (Hirt-Minkowsk P. et al., Nephron Clin Pract114:c219-c235, 2010; Goldberg R. J. et al., Am J Kidney Dis56(6):1168-1174, 2010). It has been shown that MVECs undergo apoptoticinjury when exposed in vitro to plasma from patients with TMA-relateddisorders (see Stefanescu et al., Blood Vol 112 (2):340-349, 2008; MitraD. et al., Blood 89:1224-1234, 1997). Apoptotic injury associated withTMAs has been documented in MVEC obtained from tissue biopsies (skin,bone, marrow, spleen, kidney, ileum) of such patients. It has also beenshown that apoptotic insults to MVECs reduces the levels ofmembrane-bound complement regulatory proteins in MVECs (see e.g., Mold &Morris, Immunology 102:359-364, 2001; Christmas et al., Immunology119:522, 2006).

A positive feedback loop involving terminal complement components isbelieved to be involved in the pathophysiology of TMAs includingatypical hemolytic-uremic syndrome (aHUS), and TMAs associated withcatastrophic antiphospholipid syndrome (CAPS), Degos disease, and TMAssecondary to cancer, cancer chemotherapy, autoimmunity andtransplantation, each of these conditions are known or thought to beresponsive to anti-C5 therapy with the mAb eculizumab (Chapin J. et al.,Brit. J. Hematol 157:772-774, 2012; Tsai et al., Br J Haematol162(4):558-559, 2013); Magro C. M. et al., Journal of Rare Diseases8:185, 2013).

The following experiment was carried out to analyze the ability of humanMASP-2 inhibitory antibody (OMS646) to block TMA patient plasma-mediatedinduction of apoptosis in primary human dermal MVECs in plasma samplesobtained from patients suffering from aHUS, ADAMTS13 deficiency-relatedthrombotic thrombocytopenic purpura (TTP), CAPS and systemic Degosdisease, as well as TMAs secondary to cancer, transplantation,autoimmune disease and chemotherapy.

Methods:

An in vitro assay was carried out to analyze the efficacy of a MASP-2inhibitory antibody (OMS646) to block TMA patient plasma-mediatedinduction of apoptosis in primary human MVECs of dermal origin asdescribed in Stefanescu R. et al., Blood Vol 112 (2):340-349, 2008,which is hereby incorporated herein by reference. The plasma samplesused in this assay were obtained from a collection of healthy controlsubjects and from individuals with either acute-phase or convalescentthrombotic microangiopathies. The presence of microangiopathy in the TMApatients was assessed by detecting schistocytes on a peripheral bloodsmear. In addition, TTP was diagnosed as described in Stefanescu R. etal., Blood Vol 112 (2):340-349, 2008.

Endothelial Cell (EC) Culture

As described in Stefanescu et al., primary human MVECs of dermal originwere purchased from ScienCell Research Labs (San Diego, Calif.). MVECsexpressed CD34 up through passages 5 and 6 (Blood 89:1224-1234, 1997).The MVECs were maintained in polystyrene flasks coated with 0.1% gelatinin water in ECM1001 medium (ScienCell Research Labs) containingendothelial cell growth supplement, penicillin, streptomycin and 15%fetal bovine serum. All MVECs were used in passages 2 to 6. Subculturesinvolved a 5 to 10 minute exposure to 0.25% trypsin-EDTA.

Apoptosis Assay

Representative primary human MVECs of dermal origin known to besusceptible to TTP/HUS plasma-induced apoptosis were washed withphosphate buffered saline (PBS) and plated in chambers of 12-wellplates, coated with 0.1% gelatin in water at 0.15×10⁶ viable cells/mL.The plated MVEC cells were starved in complete media for 24 hours thenexposed to varying concentrations (2% to 20% v/v) of TMA patient plasmasamples or healthy donor plasma for 18 hours in the presence or absenceof MASP-2 mAb OMS646 (150 μg/mL) and the cells were then harvested bytrypsinization. Each TMA patient sample was analyzed in duplicate. Thedegree of plasma-mediated apoptosis was assessed using propidium iodide(PI) staining, with >5×10³ cells analyzed in a cytofluorograph and A0peaks defined by computer software (MCycle Av, Phoenix Flow Systems, SanDiego, Calif.). Enzyme-linked immunosorbent assay (ELISA)-basedquantitation of cytoplasmic histone-associated DNA fragments from celllysate was also performed as per the manufacturer's directions (RocheDiagnostics, Mannheim, Germany).

Results:

The results of the TMA patient plasma-induced MVEC apoptosis assay inthe presence of MASP-2 mAb (OMS646) are shown below in Table 27.

TABLE 27 TMA patient plasma tested on primary human MVEC of dermalorigin in the presence of MASP-2 mAb (OMS646) Clinical DiagnosisDiagnosis (TMA) Diagnosis based on protection Subject Age/ and otherMASP-2 based on ADAMS ADAMS with # Sex conditions ng/ml Cre/LDH C5asC5-b9 Activity activity OMS646  #2 41/f TTP 174 TTP 34.42 772   30%aHUS responder  #3 52/f TTP 150 TTP 48.32 1399   70% aHUS non- responder #4 20/m TTP 224 TTP 36.9 1187 <10% TTP responder #10 60/f TTP 175.4 TTP49.5 4406   64% aHUS non- responder #11 59/f TTP 144.9 TTP 40.3 1352<10% TTP non- responder #13 49/f HUS, 142.8 TTP 48.6 3843   86% aHUSnon- Cancer, responder TTP #42 27/m TTP 341.5 TTP 100.0 5332  <5% TTPnon- responder #46 25/f TTP, 225.11 TTP 53.9 3426 ND ND responder Degos,SLE #48 53/f TTP, 788.5 aHUS 31.2 1066   66% aHUS responder SLE,nephritis s/p renal transplant #49 64/f TTP, 494.5 35.4 2100 ND NDresponder APLAs, CVA #51 25/f aHUS, 313.1 TTP 26.8 1595   23% aHUSresponder APLAs #52 56/f aHUS, 333.1 TTP 18.9 1103   97% aHUS non- SLEresponder #53 56/f aHUS 189.9 Remission 28.69 344   74% aHUS non-remission TTP responder Abbreviations used in Table 27: “APLAs” =antiphospholipid antibodies, associated with Catastrophicantiphospholipid syndrome (CAPS). “SLE” = systemic lupus erythematosus“CVA” = cerebrovascular accident (stroke)

Consistent with the results reported in Stefanescu R. et al., Blood Vol112 (2):340-349, 2008, significant apoptosis was observed for primaryMVECs of dermal origin in the presence of the thirteen TMA patientplasma samples in the absence of MASP-2 antibody. Control plasma samplesfrom healthy human subjects were run in parallel and did not induceapoptosis in the MVECs (data not shown). As shown in Table 27, theMASP-2 inhibitory mAb (OMS646) inhibited TMA patient plasma-mediatedinduction of apoptosis in primary MVECs (“responders” in Table 27) in 6of the 13 patient plasma samples tested (46%). In particular, it isnoted that MASP-2 inhibitory mAb (OMS646) inhibited apoptosis in plasmaobtained from patients suffering from aHUS, TTP, Degos disease, SLE,transplant, and APLAs (CAPS). With regard to the seven patient samplestested in this assay in which the MASP-2 mAb did not block apoptosis(“non-responders” in Table 27), it is noted that apoptosis can beinduced by several pathways, not all of which are complement dependent.For example, as noted in Stefanescu R. et al., Blood Vol 112(2):340-349, 2008, apoptosis in an EC assay is dependent on the basal ECactivation state which is influenced by plasma factors which may play arole in determining the level of insult required to induce apoptosis. Asfurther noted in Stefanescu R. et al., additional factors capable ofmodulating apoptosis may be present in the TMA patient plasma, such ascytokines and various components of the complement system. Therefore,due to these complicating factors, it is not surprising that the MASP-2antibody did not show a blocking effect in all of the plasma samplesthat exhibited TMA-plasma induced apoptosis.

Further in this regard, it is noted that a similar analysis was carriedout using TMA-plasma induced apoptosis assay with the anti-C5 antibodyeculizumab and very similar results were observed (see Chapin et al.,Blood (ASH Annual Meeting Abstracts): Abstract #3342, 120: 2012).Clinical efficacy of eculizumab, a highly successful commercial product,appears greater than the efficacy demonstrated in this model, suggestingthat this in vitro model may underestimate the clinical potential ofcomplement inhibitory drugs.

These results demonstrate that a MASP-2 inhibitory antibody such asOMS646 is effective at inhibiting TMA-plasma-induced apoptosis in plasmaobtained from patients suffering from a TMA such as aHUS, TTP, Degosdisease, SLE, transplant, and APLAs (CAPS). It is known that endothelialdamage and apoptosis play a key role in the pathology of TMAs such asidiopathic TTP and sporadic HUS (Kim et al., Microvascular Research vol62(2):83-93, 2001). As described in Dang et al., apoptosis wasdemonstrated in the splenic red pulp of TTP patients but not in healthycontrol subjects (Dang et al., Blood 93(4):1264-1270, 1999). Evidence ofapoptosis has also been observed in renal glomerular cells of MVECorigin in an HUS patient (Arends M. J. et al., Hum Pathol 20:89, 1989).Therefore, it is expected that administration of a MASP-2 inhibitoryagent, such as a MASP-2 inhibitory antibody (e.g., OMS646) will be aneffective therapy in patients suffering from a TMA such as aHUS, TTPorother microangiopathic disorder such as other TMAs including CAPS,systemic Degos disease, and a TMA secondary to cancer; a TMA secondaryto chemotherapy, or a TMA secondary to transplantation.

In accordance with the foregoing, in one embodiment, the inventionprovides a method of inhibiting endothelial cell damage and/orendothelial cell apoptosis, and/or thrombus formation in a subjectsuffering from, or at risk for developing, a thrombotic microangiopathy(TMA) comprising administering to the subject a composition comprisingan amount of a MASP-2 inhibitory antibody effective to inhibitMASP-2-dependent complement activation. In one embodiment, the TMA isselected from the group consisting of atypical hemolytic uremic syndrome(aHUS), thrombotic thrombocytopenic purpura (TTP) and hemolytic uremicsyndrome (HUS). In one embodiment, the TMA is aHUS. In one embodiment,the composition is administered to an aHUS patient during the acutephase of the disease. In one embodiment, the composition is administeredto an aHUS patient during the remission phase (i.e., in a subject thathas recovered or partially recovered from an episode of acute phaseaHUS, such remission evidenced, for example, by increased platelet countand/or reduced serum LDH concentrations, for example as described inLoirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, herebyincorporated herein by reference). In one embodiment, the subject issuffering from, or at risk for developing a TMA that is (i) a TMAsecondary to cancer; (ii) a TMA secondary to chemotherapy; or (iii) aTMA secondary to transplantation (e.g., organ transplantation, such askidney transplantation or allogeneic hematopoietic stem celltransplantation). In one embodiment, the subject is suffering from, orat risk for developing Upshaw-Schulman Syndrome (USS). In oneembodiment, the subject is suffering from, or at risk for developingDegos disease. In one embodiment, the subject is suffering from, or atrisk for developing Catastrophic Antiphospholipid Syndrome (CAPS).

In accordance with any of the disclosed embodiments herein, the MASP-2inhibitory antibody exhibits at least one or more of the followingcharacteristics: said antibody binds human MASP-2 with a K_(D) of 10 nMor less, said antibody binds an epitope in the CCP1 domain of MASP-2,said antibody inhibits C3b deposition in an in vitro assay in 1% humanserum at an IC₅₀ of 10 nM or less, said antibody inhibits C3b depositionin 90% human serum with an IC₅₀ of 30 nM or less, wherein the antibodyis an antibody fragment selected from the group consisting of Fv, Fab,Fab′, F(ab)₂ and F(ab′)₂, wherein the antibody is a single-chainmolecule, wherein said antibody is an IgG2 molecule, wherein saidantibody is an IgG1 molecule, wherein said antibody is an IgG4 molecule,wherein the IgG4 molecule comprises a S228P mutation, and/or wherein theantibody does not substantially inhibit the classical pathway. In oneembodiment, the antibody binds to MASP-2 and selectively inhibits thelectin pathway and does not substantially inhibit the alternativepathway. In one embodiment, the antibody binds to MASP-2 and selectivelyinhibits the lectin pathway and does not substantially inhibit theclassical pathway (i.e., inhibits the lectin pathway while leaving theclassical complement pathway intact).

In one embodiment, the MASP-2 inhibitory antibody inhibits plasmainduced MVEC apoptosis in serum from a subject suffering from a TMA suchas aHUS (acute or remission phase), hemolytic uremic syndrome (HUS),thrombotic thrombocytopenic purpura (TTP), a TMA secondary to cancer; aTMA secondary to chemotherapy; a TMA secondary to transplantation (e.g.,organ transplantation, such as kidney transplantation or allogeneichematopoietic stem cell transplantation), or in serum from a subjectsuffering from Upshaw-Schulman Syndrome (USS), or in serum from asubject suffering from Degos disease, or in a subject suffering fromCatastrophic Antiphospholipid Syndrome (CAPS), wherein the plasmainduced MVEC apoptosis is inhibited by at least 5%, such as at least10%, such as at least 20%, such as at least 30%, such as at least 40%,such as at least 50%, such as at least 60%, such as at least 70%, suchas at least 80% such as at least 85%, such as at least 90%, such as atleast 95% up to 99%, as compared to untreated serum. In someembodiments, the MASP-2 inhibitory antibody inhibits thrombus formationin serum from a subject suffering from a TMA (e.g., such as aHUS (acuteor remission phase), hemolytic uremic syndrome (HUS), thromboticthrombocytopenic purpura (TTP), a TMA secondary to cancer; a TMAsecondary to chemotherapy; a TMA secondary to transplantation (e.g.,organ transplantation, such as kidney transplantation or allogeneichematopoietic stem cell transplantation), or in serum from a subjectsuffering from Upshaw-Schulman Syndrome (USS), or in serum from asubject suffering from Degos disease, or in a subject suffering fromCatastrophic Antiphospholipid Syndrome (CAPS)), at a level of at least20 percent or greater, (such as at least 30%, at least 40%, at least50%) more than the inhibitory effect on C5b-9 deposition in serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to thesubject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibitingthrombus formation in a subject suffering from a TMA (such as aHUS(acute or remission phase), hemolytic uremic syndrome (HUS), thromboticthrombocytopenic purpura (TTP), a TMA secondary to cancer; a TMAsecondary to chemotherapy; a TMA secondary to transplantation (e.g.,organ transplantation, such as kidney transplantation or allogeneichematopoietic stem cell transplantation), or in serum from a subjectsuffering from Upshaw-Schulman Syndrome (USS), or in serum from asubject suffering from Degos disease, or in a subject suffering fromCatastrophic Antiphospholipid Syndrome (CAPS)), comprising administeringto the subject a composition comprising an amount of a MASP-2 inhibitoryantibody, or antigen binding fragment thereof, comprising (I) (a) aheavy-chain variable region comprising: i) a heavy-chain CDR-H1comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) aheavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acidsequence from 95-102 of SEQ ID NO:67 and b) a light-chain variableregion comprising: i) a light-chain CDR-L1 comprising the amino acidsequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii)a light-chain CDR-L3 comprising the amino acid sequence from 89-97 ofSEQ ID NO:70, or (II) a variant thereof comprising a heavy-chainvariable region with at least 90% identity to SEQ ID NO:67 (e.g., atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to SEQ IDNO:67) and a light-chain variable region with at least 90% identity(e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% identity toSEQ ID NO:70.

In one embodiment, the subject is suffering from a TMA selected from thegroup consisting of a TMA secondary to cancer; a TMA secondary tochemotherapy; a TMA secondary to transplantation (e.g., organtransplantation, such as kidney transplantation or allogeneichematopoietic stem cell transplantation), Upshaw-Schulman Syndrome(USS), Degos disease, and Catastrophic Antiphospholipid Syndrome (CAPS).

In some embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a heavy-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:67. Insome embodiments, the method comprises administering to the subject acomposition comprising an amount of a MASP-2 inhibitory antibody, orantigen binding fragment thereof, comprising a light-chain variableregion comprising the amino acid sequence set forth as SEQ ID NO:70.

In some embodiments, the method comprises administering to the subject acomposition comprising a MASP-2 inhibitory antibody, or antigen bindingfragment thereof, that specifically recognizes at least part of anepitope on human MASP-2 recognized by reference antibody OMS646comprising a heavy-chain variable region as set forth in SEQ ID NO:67and a light-chain variable region as set forth in SEQ ID NO:70.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method of inhibiting MASP-2-dependent complement activation in asubject suffering from Catastrophic Antiphospholipid Syndrome (CAPS),comprising administering to the subject a composition comprising anamount of a MASP-2 inhibitory agent effective to inhibitMASP-2-dependent complement activation.
 2. The method of claim 1,wherein the MASP-2 inhibitory agent is an anti-MASP-2 antibody orfragment thereof.
 3. The method of claim 2, wherein the MASP-2inhibitory agent is an anti-MASP-2 monoclonal antibody, or fragmentthereof that specifically binds to a portion of SEQ ID NO:6.
 4. Themethod of claim 2, wherein the subject has previously undergone, or iscurrently undergoing, treatment with a terminal complement inhibitorthat inhibits cleavage of complement protein C5.
 5. The method of claim2, wherein the method further comprises administering to the subject aterminal complement inhibitor that inhibits cleavage of complementprotein C5.
 6. The method of claim 5, wherein the terminal complementinhibitor is a humanized anti-C5 antibody or antigen-binding fragmentthereof.
 7. The method of claim 5, wherein the terminal complementinhibitor is eculizumab.
 8. The method of claim 2, wherein the antibodyor fragment thereof is selected from the group consisting of arecombinant antibody, an antibody having reduced effector function, achimeric antibody, a humanized antibody and a human antibody.
 9. Themethod of claim 1, wherein the composition is administeredsubcutaneously, intra-muscularly, intra-arterially, intravenously, or asan inhalant.
 10. The method of claim 3, wherein said MASP-2 inhibitoryantibody binds human MASP-2 with a K_(D) of 10 nM or less.
 11. Themethod of claim 3, wherein said MASP-2 inhibitory antibody binds anepitope in the CCP1 domain of MASP-2.
 12. The method of claim 3, whereinsaid MASP-2 inhibitory antibody inhibits C3b deposition in an in vitroassay in 1% human serum at an IC₅₀ of 10 nM or less.
 13. The method ofclaim 3, wherein said MASP-2 inhibitory antibody inhibits C3b depositionin 90% human serum with an IC₅₀ of 30 nM or less.
 14. The method ofclaim 3, wherein said MASP-2 inhibitory antibody is an antibody fragmentselected from the group consisting of Fv, Fab, Fab′, F(ab) and F(ab′)₂.15. The method of claim 3, wherein said MASP-2 inhibitory antibody is asingle-chain molecule.
 16. The method of claim 3, wherein said MASP-2inhibitory antibody is selected from the group consisting of an IgG 1molecule, an IgG2 and an IgG4 molecule.
 17. The method of claim 3,wherein the IgG4 molecule comprises a S228P mutation.
 18. The method ofclaim 3, wherein said MASP-2 inhibitory antibody does not substantiallyinhibit the classical pathway.
 19. The method of claim 3, wherein theMASP-2 inhibitory monoclonal antibody, or antigen-binding fragmentthereof, comprises: (a) a heavy-chain variable region comprising: i) aheavy chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acidsequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3comprising the amino acid sequence from 95-102 of SEQ ID NO:67 and (b) alight-chain variable region comprising: i) a light-chain CDR-L1comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) alight-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQID NO:70; and iii) a light-chain CDR-L3 comprising the amino acidsequence from 89-97 of SEQ ID NO:70.
 20. The method of claim 3, whereinthe MASP-2 inhibitory monoclonal antibody comprises a heavy-chainvariable region set forth as SEQ ID NO:67 and a light-chain variableregion set forth as SEQ ID NO:70.